Compounds for nonsense suppression and methods for their use

ABSTRACT

The present invention relates to methods, compounds, and compositions for treating or preventing diseases associated with nonsense mutations in an mRNA by administering the compounds or compositions of the present invention. More particularly, the present invention relates to methods, compounds, and compositions for suppressing premature translation termination associated with a nonsense mutation in an mRNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/577,192, filed Apr. 12, 2007, which U.S. application Ser. No.11/577,192 is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2005/037052, filed Oct. 13, 2005,which claims priority to and the benefit under 35 U.S.C. §119 of U.S.Provisional Application No. 60/617,670, filed Oct. 13, 2004, U.S.Provisional Application No. 60/617,634, filed Oct. 13, 2004, U.S.Provisional Application No. 60/617,633, filed Oct. 13, 2004, U.S.Provisional Application No. 60/617,655, filed Oct. 13, 2004, U.S.Provisional Application No. 60/617,653, filed Oct. 13, 2004, and U.S.Provisional Application No. 60/624,170, filed Nov. 3, 2004, the entirecontents of all of which applications are incorporated herein byreference.

RELATED APPLICATIONS

The present application also incorporates by reference herein in theirentireties International Application No. PCT/US2005/036673, filed Oct.13, 2005, International Application No. PCT/US2005/036761, filed Oct.13, 2005, International Application No. PCT/US2005/036762, filed Oct.13, 2005, and International Application No. PCT/US 2005/036764, filedOct. 13, 2005.

YIELD OF THE INVENTION

The present invention relates to methods, compounds, and compositionsfor treating or preventing diseases associated with nonsense mutationsin an mRNA by administering the compounds or compositions of the presentinvention. More particularly, the present invention relates to methodsand compounds and compositions for suppressing premature translationtermination associated with a nonsense mutation in an mRNA.

BACKGROUND OF THE INVENTION

Gene expression in cells depends upon the sequential processes oftranscription and translation. Together, these processes produce aprotein from the nucleotide sequence of its corresponding gene.

Transcription involves the synthesis of mRNA from DNA by RNA polymerase.Transcription begins at a promoter region of the gene and continuesuntil termination is induced, such as by the formation of a stem-loopstructure in the nascent RNA or the binding of the rho gene product.

Protein is produced from mRNA by the process of translation, occurringon the ribosome with the aid of tRNA, tRNA synthetases and various otherprotein and RNA species. Translation comprises the three phases ofinitiation, elongation and termination. Translation is initiated by theformation of an initiation complex consisting of protein factors, mRNA,tRNA, cofactors and the ribosomal subunits that recognize signals' onthe mRNA that direct the translation machinery to begin translation onthe mRNA. Once the initiation complex is formed, growth of thepolypeptide chain occurs by the repetitive addition of amino acids bythe peptidyl transferase activity of the ribosome as well as tRNA andtRNA synthetases. The presence of one of the three termination codons(UAA, UAG, UGA) in the A site of the ribosome signals the polypeptidechain release factors (RFs) to bind and recognize the terminationsignal. Subsequently, the ester bond between the 3′ nucleotide of thetRNA located in the ribosome's P site and the nascent polypeptide chainis hydrolyzed, the completed polypeptide chain is released, and theribosome subunits are recycled for another round of translation.

Mutations of the DNA sequence in which the number of bases is alteredare categorized as insertion or deletion mutations (e.g., frameshiftmutations) and can result in major disruptions of the genome. Mutationsof the DNA that change one base into another and result in an amino,acid substitution are labeled missense mutations. Base substitutions aresubdivided into the classes of transitions (one purine to anotherpurine, or one pyrimidine to another pyrimidine) and transversions (apurine to a pyrimidine, or a pyrimidine to a purine).

Transition and transversion mutations can result in a nonsense mutationchanging an amino acid codon into one of the three stop codons. Thesepremature stop codons can produce aberrant proteins in cells as a resultof premature translation termination. A nonsense mutation in anessential gene can be lethal and can also result in a number of humandiseases, such as, cancers, lysosomal storage disorders, the musculardystrophies, cystic fibrosis and hemophilia, to name a few.

The human p53 gene is the most commonly mutated gene in human cancer(Zambetti, G. P. and Levine, A., FASEB 7:855-865 (1993)). Found in bothgenetic and spontaneous cancers, over 50 different types of humancancers contain p5.3 mutations and mutations of this gene occur in50-55% of all human cancers (Hollstein, M., et al., Nucleic Acids Res.22:3551-55 (1994); International Agency for Research on Cancer (IARC)database). Approximately 70% of colorectal cancer, 50% of lung cancerand 40% of breast cancers contain mutant p53 (Koshland, D., Science262:1953 (1993)). Aberrant forms of p53 are associated with poorprognosis, more aggressive tumors, metastasis, and lower 5 year survivalrates (Id.). p53's role in the induction of cell growth arrest and/orapoptosis upon DNA damage is believed to be essential for thedestruction of mutated cells that would have otherwise gained a growthadvantage, in addition, p53 sensitizes rapidly dividing cells toapoptotic signals. Of greater than 15,000 reported mutations in the p53gene, approximately 7% are nonsense mutations. Accordingly, there is aneed for a safe and effective treatment directed to p53 nonsensemutations.

In bacterial and eukaryotic strains with nonsense mutations, suppressionof the nonsense mutation can arise as a result of a mutation in one ofthe tRNA molecules so that the mutant tRNA can recognize the nonsensecodon, as a result of mutations in proteins that are involved in thetranslation process, as a result of mutations in the ribosome (eitherthe ribosomal RNA or ribosomal proteins), or by the addition ofcompounds known to alter the translation process (for example,cycloheximide or the aminoglycoside antibiotics). The result is that anamino acid will be incorporated into the polypeptide chain, at the siteof the nonsense mutation, and translation will not prematurely terminateat the nonsense codon. The inserted amino acid will not necessarily beidentical to the original amino acid of the wild-type protein, however,many amino acid substitutions do not have a gross effect on proteinstructure or function. Thus, a protein produced by the suppression of anonsense mutation would be likely to possess activity close to that ofthe wild-type protein. This scenario provides an opportunity to treatdiseases associated with nonsense mutations by avoiding prematuretermination of translation through suppression of the nonsense mutation.

The ability of aminoglycoside antibiotics to promote read-through ofeukaryotic stop codons has attracted interest in these drugs aspotential therapeutic agents in human diseases caused by nonsensemutations. One disease for which such a therapeutic strategy may beviable is classical late infantile neuronal ceroid lipofuscinosis(LINCL), a fatal childhood neurodegenerative disease with currently noeffective treatment. Premature stop codon mutations in the gene CLN2encoding the lysosomal tripeptidyl-peptidase 1 (TPP-I) are associatedwith disease in approximately half of children diagnosed with LINCL. Theability of the aminoglycoside gentamicin to restore TPP-1 activity inLINCL, cell lines has been examined. In one patient-derived cell linethat was compound heterozygous for a commonly seen nonsense mutation(Arg208Stop) and a different rare nonsense mutation, approximately 7% ofnormal levels of TPP-I were maximally restored with gentamicintreatment. These results suggest that pharmacological suppression ofnonsense mutations by aminoglycosides or functionally similarpharmaceuticals may have therapeutic potential in LINCL (Sleat et. al.,Eur. J. Ped. Neurol. 5:Suppl A 57-62 (2000).

In cultured cells having premature stop codons in the Cystic FibrosisTransmembrane Conductance Regulator (CFTR) gene, treatment withaminoglycosides led to the production of full-length CFTR (Bedwell et.al., Nat. Med. 3:1280-1284 (1997); Howard et, al. Nat. Med. 2; 467-469(1996)). In mouse models for Duchenne muscular dystrophy, gentamicinsulfate was observed to suppress translational termination at prematurestop codons resulting in full-length dystrophin (Barton-Davis et. al.,J. Clin. Invest. 104:375-381 (1999)). A small increase in the amount offull-length dystrophin provided protection against contraction-induceddamage in the mdx mice. The amino acid inserted at the site of thenonsense codon was not determined in these studies.

Accordingly, small molecule therapeutics or prophylactics that suppresspremature translation termination by mediating the misreading of thenonsense codon would be useful for the treatment of a number ofdiseases. The discovery of small molecule drags, particularly orallybioavailable drugs, can lead to the introduction of a broad spectrum ofselective therapeutics or prophylactics to the public which can be usedagainst disease caused by nonsense mutations is just beginning.

Clitocine (6-Amino-5-nitro-4-(β-D-ribo-furanosylamino)pyrimidine) is anaturally occurring exocyclic amino nucleoside that was first isolatedfrom the mushroom Clitocybe inversa (Kubo et al., Tet. Lett. 27: 4277(1986)). The total synthesis of clitocine has also been reported. (Mosset al., J. Med. Chem., 31:786-790 (1988) and Kamikawa et al., J. Chem.Soc. Chem. Commun. 195 (1988)). Clitocine has been reported to possessinsecticidal activity and cytostatic activity against leukemia celllines (Kubo et al., Tet. Lett 27: 4277 (1986) and Moss et al., J. Med.Chem. 31:786-790 (1988)). However, the use of clitocine as a therapeuticfor diseases associated with a nonsense mutation has not been discloseduntil now. Nor has anyone reported the development of an analogue orderivative of clitocine that has utility as a therapeutic for cancer ora disease associated with a nonsense mutation.

Thus, there remains a need to develop, characterize, and optimize leadmolecules for the development of novel drags for treating or preventingdiseases associated with nonsense mutations of mRNA. Accordingly, it isan object of the present invention to provide such compounds.

All documents referred to herein are incorporated by reference into thepresent application as though fully set forth herein.

SUMMARY OF THE INVENTION

In accordance with the present invention, compounds that suppresspremature translation termination associated with a nonsense mutation inmRNA have been identified, and methods for their use provided.

In one aspect of the invention, compounds of Formula (I) are providedwhich are useful for suppressing premature translation terminationassociated with a nonsense mutation in mRNA; and for treating diseasesassociated with nonsense mutations in mRNA:

wherein:

X is a halogen;

R is a C₁-C₈ alkyl group; a C₁-C₄ haloalkyl group; an —OR₁ group; or anamino group which is optionally substituted with one or twoindependently selected R₂ groups;

R₁ is a C₁-C₈ alkyl group which is optionally substituted with one ormore independently selected R_(a) groups; a —R_(b) group; a pyrrolidinylgroup which is optionally substituted with one or more independentlyselected C₁-C₄ alkyl or oxo groups; a piperidyl group which isoptionally substituted with one or more independently selected C₁-C₄alkyl groups, benzyl groups, or carboxy groups optionally substitutedwith one or more C₁-C₄ alkyl or C₁-C₄ alkoxy groups; a tetrahydro-furylgroup; a tetrahydro-pyranyl group; a tetrahydro-naphthyl group; or anindanyl group;

R₂ is a hydrogen, a C₁-C₆ alkyl group; a C₁-C₄ haloalkyl group; a C₁-C₄alkoxy group; a —R_(b) group; a pyrimidinyl group; a pyridyl group; asulfonyl group optionally substituted with an —R_(b) group; or two R₂groups together with the amino to which they are attached form amorpholinyl group, a pyrrolidinyl group, an isoindolinyl group, or apiperazinyl group which is optionally substituted with a phenyl group;

wherein R_(a) is a halogen; a C₁-C₄ alkoxy group; a carbamoyl groupwhich is optionally substituted with one or two independently selectedC₁-C₄ alkyl or C₁-C₄ alkoxy groups; a phosphinoyl group which isoptionally substituted with one or two independently selected C₁-C₄alkyl or C₁-C₄ alkoxy groups; a morpholinyl group; a pyridyl group; or a—R_(b) group; and

wherein R_(b) is a C₆-C₈ aryl which is optionally substituted with oneor more of the following, independently selected: a hydroxy, a halogen,a C₁-C₄ alkyl group, a C₁-C₄ haloalkyl group, a C₁-C₄ alkoxy group, oran amino group which is optionally substituted with one or moreindependently selected C₁-C₄ alkyl groups;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,polymorph, racemate or stereoisomer of said compound of Formula 1.

In another aspect of the invention, methods are provided for thesuppression of premature translation termination associated with anonsense mutation, and for the prevention or treatment of diseasesassociated with nonsense mutations of mRNA. Such diseases include, butare not limited to, genetic diseases caused by premature translationtermination associated with a nonsense mutation, such as a CNS disease,an inflammatory disease, a neurodegenerative disease, an autoimmunedisease, a cardiovascular disease, or a pulmonary disease; morepreferably the disease is cancer (or other proliferative diseases),amyloidosis, Alzheimer's disease, atherosclerosis, giantism, dwarfism,hypothyroidism, hyperthyroidism, cystic fibrosis, aging, obesity,Parkinson's disease, Niemann Pick's disease, familialhypercholesterolemia, retinitis pigmentosa, Marfan syndrome, lysosomalstorage disorders, the muscular dystrophies, cystic fibrosis,hemophilia, or classical late infantile neuronal ceroid lipofuscinosis(LINCL).

In one embodiment, the invention is directed to methods for suppressingpremature translation termination associated with a nonsense mutation inmRNA comprising administering a nonsense-suppressing amount of at leastone compound of the invention to a subject in need thereof.

In yet another embodiment, methods for treating cancer, lysosomalstorage disorders, a muscular dystrophy, cystic fibrosis, hemophilia, orclassical late infantile neuronal ceroid lipofuscinosis are providedcomprising administering a therapeutically effective amount of at leastone compound of the invention to a subject in need thereof.

These and other aspects of the invention will be more clearly understoodwith reference to the following embodiments, detailed description, andclaims, for example.

Certain Embodiments

1. A method of treating or preventing a disease resulting from a somaticmutation comprising administration to a patient in need thereof aneffective amount of a compound of Formula 1:

wherein:

X is a halogen;

R is a C₁-C₈ alkyl group; a C₁-C₄ haloalkyl group; an —OR₁ group; or anamino group which is optionally substituted with one or twoindependently selected R₂ groups;

R₁ is a C₁-C₈ alkyl group which is optionally substituted with one ormore independently selected R_(a) groups; a —R_(b) group; a pyrrolidinylgroup which is optionally substituted with one or more independentlyselected C₁-C₄ alkyl or oxo groups; a piperidyl group which isoptionally substituted with one or more independently selected C₁-C₄alkyl groups, benzyl groups, or carboxy groups optionally substitutedwith one or more C₁-C₄ alkyl or C₁-C₄ alkoxy groups; a tetrahydro-furylgroup; a tetrahydro-pyranyl group; a tetrahydro-naphthyl group; or anindanyl group;

R₂ is a hydrogen, a C₁-C₆ alkyl group; a C₁-C₄ haloalkyl group; a C₁-C₄alkoxy group; a —R_(b) group; a pyrimidinyl group; a pyridyl group; asulfonyl group optionally substituted with an —R_(b) group; or two R₂groups together with the amino to which they are attached form amorpholinyl group, a pyrrolidinyl group, an isoindolinyl group, or apiperazinyl group which is optionally substituted with a phenyl group;

wherein R_(a) is a halogen; a C₁-C₄ alkoxy group; a carbamoyl groupwhich is optionally substituted with one or two independently selectedC₁-C₄ alkyl or C₁-C₄ alkoxy groups; a phosphinoyl group which isoptionally substituted with one or two independently selected C₁-C₄alkyl or C₁-C₄ alkoxy groups; a morpholinyl group; a pyridyl group; oran —R_(b) group; and

wherein R_(b) is a C₆-C₈ aryl which is optionally substituted with oneor more of the following, independently selected: a hydroxy, a halogen,a C₁-C₄ alkyl group, a C₁-C₄ haloalkyl group, a C₁-C₄ alkoxy group, oran amino group which is optionally substituted with one or moreindependently selected C₁-C₄ alkyl groups;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,polymorph, racemate or stereoisomer of said compound of Formula 1.

2. The method of embodiment 1, wherein the compound of Formula 1, or apharmaceutically acceptable salt, hydrate, solvate, clathrate polymorph,racemate or stereoisomer thereof, is administered as a compositioncomprising the compound and a pharmaceutically acceptable carrier ordiluent.3. The method of embodiment 1, wherein the administration isintravenous.4. The method of embodiment 1, wherein X is fluorine and is in an orthoposition.5. The method of embodiment 1, wherein the compound of Formula 1 is acompound of Formula 1A:

6. The method of embodiment 5, wherein R₁ is a C₁-C₈ alkyl group whichis optionally substituted with one or more independently selected R_(a)groups.7. The method of embodiment 5, wherein X is fluorine and is located inan ortho position.8. The method of embodiment 1, wherein the compound of Formula 1 is acompound of Formula 1B:

9. The method of embodiment 8, wherein at least one R₂ is H.10. The method of embodiment 8, wherein X is fluorine and is located inan ortho position.11. The method of embodiment 1, wherein the compound of Formula 1 is acompound of Formula 1C:

wherein R_(c) is independently selected from hydrogen, a halogen, amethyl, or an ethyl.

12. The method of embodiment 11, wherein at least one R_(c) is fluorine.

13. The method of embodiment 11, wherein X is fluorine and is located inan ortho position.

14. The method of embodiment 1, where R is a C₁-C₄ alkyl group; a C₁-C₄haloalkyl group; an —OR₁, group; or an amino group which is optionallysubstituted with one or two independently selected R₂ groups.

15. A method of treating or preventing an autoimmune disease, a blooddisease, a collagen disease, diabetes, a neurodegenerative disease, acardiovascular disease, a pulmonary disease, or an inflammatory diseaseor central nervous system disease comprising administration to a patientin need thereof an effective amount of a compound of Formula 1, or apharmaceutically acceptable salt, hydrate, solvate, clathrate, racemateor stereoisomer thereof.16. The method of embodiment 15, wherein the administration isintravenous.17. The method of embodiment 15, wherein the autoimmune disease isrheumatoid arthritis or graft versus host disease.18. The method of embodiment 15, wherein the inflammatory disease isarthritis.19. The method of embodiment 15, wherein the central nervous systemdisease is multiple sclerosis, muscular dystrophy, Duchenne musculardystrophy, Alzheimer's disease, a neurodegenerative disease orParkinson's disease.20. The method of embodiment 15, wherein the blood disorder ishemophilia, Von Willebrand disease, ataxia-telangiectasia, β-thalassemiaor kidney stones.21. The method of embodiment 15, wherein the collagen disease isosteogenesis imperfecta or cirrhosis.22. A method of treating or preventing familial polycythemia,immunodeficiency, kidney disease, kidney stones, heart disease,ataxia-telangiectasia, cystic fibrosis, muscular dystrophy, familialhypercholesterolemia, retinitis pigmentosa, amyloidosis, hemophilia,Alzheimer's disease, Tay Sachs disease, Niemann Pick disease,Parkinson's disease, atherosclerosis, giantism, dwarfism,hypothyroidism, hyperthyroidism, aging, obesity, Duchenne musculardystrophy, epidermolysis bullosa or Marfan syndrome comprisingadministration to a patient in need thereof an effective amount of acompound of Formula 1, or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, racemate or stereoisomer thereof.23. The method of embodiment 22, wherein the administration isintravenous.24. A method of treating or preventing cancer in a human comprisingadministration to a human in need thereof an effective amount of acompound of Formula 1, or a pharmaceutically acceptable salt, hydrate,solvate, clathrate, racemate or stereoisomer thereof.25. The method of embodiment 24, wherein the administration isintravenous.26. The method of embodiment 24, wherein the cancer is of the head andneck, eye, skin, mouth, throat, esophagus, chest, bone, blood, lung,colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney,liver, pancreas, brain, intestine, heart or adrenals.27. The method of embodiment 24, wherein the compound, or apharmaceutically acceptable salt, hydrate, solvate, clathrate orstereoisomer thereof, comprises a pharmaceutically acceptable carrier ordiluent.28. The method of embodiment 24, wherein the cancer is a solid tumor,29. The method of embodiment 24, wherein the cancer is sarcoma,carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,menangioma, melanoma, neuroblastoma, retinoblastoma, a blood-born tumoror multiple myeloma.30. The method of embodiment 24, wherein the cancer is acutelymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute monoblastic leukemia, acuteerythroleukemic leukemia, acute megakaryoblastic leukemia, acutemyelomonocytic leukemia, acute nonlymphocyctic leukemia, acuteundifferentiated leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, hairy cell leukemia, or multiple myeloma.31. A method of treating or preventing a disease associated with amutation of the p53 gene comprising administration to a patient in needthereof an effective amount of a compound of Formula 1, or apharmaceutically acceptable salt, hydrate, solvate, clathrate, racemateor stereoisomer thereof.32. The method of embodiment 31, wherein the administration isintravenous.33. The method of embodiment 31, wherein the disease is sarcoma,carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,menangioma, melanoma, neuroblastoma, retinoblastoma a blood-born tumoror multiple myeloma.34. A method of inhibiting the growth of a cancer cell comprisingcontacting the cancer cell with an effective amount of a compound ofFormula 1, or a pharmaceutically acceptable salt, hydrate, solvate,clathrate, polymorph, racemate or stereoisomer thereof,35. A method for selectively producing a protein in a mammal comprising,transcribing a gene containing a nonsense mutation in the mammal; andproviding an effective amount of a compound of the present invention tosaid mammal, wherein said protein is produced from said gene containinga nonsense mutation.36. A compound of Formula 1:

wherein:

-   -   X is a halogen;    -   R is a C₁-C₈ alkyl group; a C₁-C₄ haloalkyl group; an —OR₁        group; or an amino group which is optionally substituted with        one or two independently selected R₂ groups;    -   R₁ is a C₁-C₈ alkyl group which is optionally substituted with        one or more independently selected R_(a) groups; a —R_(b) group;        a pyrrolidinyl group which is optionally substituted with one or        more independently selected C₁-C₄ alkyl or oxo groups; a        piperidyl group which is optionally substituted with one or more        independently selected C₁-C₄ alkyl groups, benzyl groups, or        carboxy groups optionally substituted with one or more C₁-C₄        alkyl or C₁-C₄ alkoxy groups; a tetrahydro-furyl group; a        tetrahydro-pyranyl group; a tetrahydro-naphthyl group; or an        indanyl group;    -   R₂ is a hydrogen, a C₁-C₆ alkyl group; a C₁-C₄ haloalkyl group;        a C₁-C₄ alkoxy group; a —R_(b) group; a pyrimidinyl group; a        pyridyl group; a sulfonyl group optionally substituted with an        —R_(b) group; or two R₂ groups together with the amino to which        they are attached form a morpholinyl group, a pyrrolidinyl        group, an isoindolinyl group, or a piperazinyl group which is        optionally substituted with a phenyl group;    -   wherein R_(a) is a halogen; a C₁-C₄ alkoxy group; a carbamoyl        group which is optionally substituted with one or two        independently selected C₁-C₄ alkyl or C₁-C₄ alkoxy groups; a        phosphinoyl group which is optionally substituted with one or        two independently selected C₁-C₄ alkyl or C₁-C₄ alkoxy groups; a        morpholinyl group; a pyridyl group; or an —R_(b) group; and    -   wherein R_(b) is a C₆-C₈ aryl which is optionally substituted        with one or more of the following, independently selected: a        hydroxy, a halogen, a C₁-C₄ alkyl group, a C₁-C₄ haloalkyl        group, a C₁-C₄ alkoxy group, or an amino group which is        optionally substituted with one or more independently selected        C₁-C₄ alkyl groups;    -   or a pharmaceutically acceptable salt, hydrate, solvate,        clathrate, polymorph, racemate or stereoisomer of said compound        of Formula 1.        37. The compound of embodiment 36, where R is a C₁-C₄ alkyl        group; a C₁-C₄ haloalkyl group; an —OR₁ group; or an amino group        which is optionally substituted with one or two independently        selected R₂ groups.        38. The compound of embodiment 36, wherein said compound is        selected from compounds 1-51.        39. A compound having the formula (Compound NO: 1):

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides schematic representations of constructs for luciferasebased assays to evaluate the suppression of a nonsense mutation.

FIG. 2 provides schematic representations of the luciferase constructsengineered to harbor one or more epitope tags in the of the luciferaseprotein.

FIG. 3 provides schematic representations of constructs for luciferasebased assays to evaluate readthrough efficiency.

DETAILED DESCRIPTION OF THE INVENTION

Premature translation termination can produce aberrant proteins whichcan be lethal or can cause a number of diseases, including asnon-limiting examples, cancers, lysosomal storage disorders, themuscular dystrophies, cystic fibrosis, and hemophilia. In accordancewith the present invention, compounds that suppress nonsense mutationshave been identified, and methods for their use provided.

A. Compounds of the Invention

In one aspect of the invention, compounds of the invention are providedwhich are useful in suppression of a nonsense mutation. Compounds of thepresent invention are also useful for increasing the expression of aprotein. In certain embodiments, the compounds of the inventionspecifically suppresses a nonsense mutation, while in other embodiments,the compounds of the invention suppress a nonsense mutation as well astreat a disease, including as non-limiting examples, cancers, lysosomalstorage disorders, the muscular dystrophies, cystic fibrosis andhemophilia.

Preferred compounds of the present invention useful in the suppressionof a nonsense mutation include those of Formula (I) as shown below.

wherein;

X is a halogen;

R is a C₁-C₈ alkyl group; a C₁-C₄ haloalkyl group; an —OR₁ group; or anamino group which is optionally substituted with one or twoindependently selected R₂ groups;

R₁ is a C₁-C₈ alkyl group which is optionally substituted with one ormore independently selected R_(a) groups; a —R_(b) group; a pyrrolidinylgroup which is optionally substituted with one or more independentlyselected C₁-C₄ alkyl or oxo groups; a piperidyl group which isoptionally substituted with one or more independently selected C₁-C₄alkyl groups, benzyl groups, or carboxy groups optionally substitutedwith one or more C₁-C₄ alkyl or C₁-C₄ alkoxy groups; a tetrahydro-furylgroup; a tetrahydro-pyranyl group; a tetrahydro-naphthyl group; or anindanyl group;

R₂ is a hydrogen, a C₁-C₆ alkyl group; a C₁-C₄ haloalkyl group; a C₁-C₄alkoxy group; a —R_(b) group; a pyrimidinyl group; a pyridyl group; asulfonyl group optionally substituted with an —R_(b) group; or two R₂groups together with the amino to which they are attached form amorpholinyl group, a pyrrolidinyl group, an isoindolinyl group, or apiperazinyl group which is optionally substituted with a phenyl group;

wherein R_(a) is a halogen; a C₁-C₄ alkoxy group; a carbamoyl groupwhich is optionally substituted with one or two independently selectedC₁-C₄ alkyl or C₁-C₄ alkoxy groups; a phosphinoyl group which isoptionally substituted with one or two independently selected C₁-C₄alkyl or C₁-C₄ alkoxy groups; a morpholinyl group; a pyridyl group; oran —R_(b) group; and

wherein R_(b) is a C₆-C₈ aryl which is optionally substituted with oneor more of the following, independently selected: a hydroxy, a halogen,a C₁-C₄ alkyl group, a C₁-C₄ haloalkyl group, a C₁-C₄ alkoxy group, oran amino group which is optionally substituted with one or moreindependently selected C₁-C₄ alkyl groups;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,polymorph, racemate or stereoisomer of said compound of Formula 1.

Preferred compounds of the present invention useful in the suppressionof a nonsense mutation also include those of Formula (1) as abovewherein:

X is a halogen;

R is a C₁-C₈ alkyl group; a C₁-C₄ haloalkyl group; an —OR₁ group; or anamino group which is optionally substituted with one or twoindependently selected R₂ groups;

R₁ is a C₁-C₈ alkyl group which is optionally substituted with one ormore independently selected R_(a) groups; a —R_(b) group; a pyrrolidinylgroup which is optionally substituted with one or more independentlyselected C₁-C₄ alkyl or oxo groups; a piperidyl group which isoptionally substituted with one or more independently selected C₁-C₄alkyl groups, benzyl groups, or carboxy groups optionally substitutedwith one or more C₁-C₄ alkyl; a tetrahydro-furyl group; atetrahydro-pyranyl group; a tetrahydro-naphthyl group; or an indanylgroup;

R₂ is a C₁-C₆ alkyl group; a C₁-C₄ haloalkyl group; a C₁-C₄ alkoxygroup; a —R_(b) group; a pyrimidinyl group; a pyridyl group; a sulfonylgroup optionally substituted with an —R_(b) group; or two R₂ groupstogether with the amino to which they are attached form a morpholinylgroup, a pyrrolidinyl group, or an isoindolinyl group;

wherein R_(a) is a halogen; a C₁-C₄ alkoxy group; a carbamoyl group; aphosphinoyl group which is optionally substituted with one or twoindependently selected C₁-C₄ alkyl or C₁-C₄ alkoxy groups; a morpholinylgroup; a pyridyl group; or a —R_(b) group; and

wherein R_(b) is a C₆-C₈ aryl which is optionally substituted with oneor more of the following, independently selected: a halogen, a C₁-C₄alkyl group, or a C₁-C₄ haloalkyl group;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,polymorph, racemate or stereoisomer of said compound of Formula 1.

With respect to Formula 1, the position of X (e.g., ortho, meta, orpara) on the phenyl ring is stated relative to the oxadiazole ring ofFormula 1.

In an embodiment of Formula 1, X is fluorine. In another embodiment ofFormula 1, X is a halogen other than fluorine. In a further embodimentof Formula 1, X is chlorine. In a further embodiment of Formula 1, X isbromine. In another embodiment of Formula 1, X is iodine.

In an embodiment of Formula 1, R is a C₁-C₈ alkyl group or a C₁-C₄haloalkyl group. In an embodiment of Formula 1, R is a C₁-C₈ alkyl groupor a C₁-C₄ haloalkyl group, and X is a halogen selected from the groupconsisting of fluorine, chlorine, bromine or iodine. In anotherembodiment of Formula 1, R is a C₁-C₈ alkyl group, or a C₁-C₄ haloalkylgroup, and X is fluorine.

A variety of embodiments of compounds of Formula 1A are consideredwithin the scope of this invention. In an embodiment of Formula 1A, R₁is a linear or branched C₁-C₈ alkyl group. In another embodiment, R₁ isa C₃-C₈ cyclic alkyl group. In a further embodiment of Formula 1A, R₁ isa C₁-C₈ alkyl group substituted with one or more R_(a) groups, whereR_(a) is an alkoxy group or a phosphinoyl group which is optionallysubstituted with one or two independently selected C₁-C₄ alkyl or C₁-C₄alkoxy groups. In another embodiment of Formula 1A, R₁ is a C₁-C₄ alkylgroup optionally substituted with one or more R_(a) groups, where R_(a)is a morpholinyl or a pyridinyl group. In an embodiment of Formula 1A,R₁ is selected from a tetrahydro-furyl group, a tetrahydro-pyranylgroup, a tetrahydro-naphthyl group, or an indanyl group. In anotherembodiment of Formula 1A, R₁ is R_(b), where R_(b) is a C₆ aryl group,optionally substituted with one or more groups selected from: hydroxy,halogen, C₁-C₄ alkyl group, haloalkyl group, C₁-C₄ alkoxy group, or anamino group which is optionally substituted with one or moreindependently selected C₁-C₄ alkyl groups. In addition, in anotherembodiment of compounds of Formula 1A, R₁ is a C₁-C₄ alkyl groupoptionally substituted with one or more independently selected R_(a)groups, where R_(a) is an R_(b) group. In an embodiment of Formula 1A, Xmay be selected from the group consisting of fluorine, chlorine, bromineand iodine. In another embodiment of Formula 1A, X may be fluorine.

A variety of embodiments of compounds of Formula 1B are envisioned. Forexample, in an embodiment of Formula 1B, the N(R₂)₂ functionality is anamino group and both R₂ groups are hydrogen. In another embodiment ofFormula 1B, neither R₂ groups is hydrogen. In an alternative embodimentof Formula 1B, only one R₂ group is hydrogen. In a further embodiment ofFormula 1B, both R₂ groups, together with the amino to which they areattached, form a morpholinyl group, a pyrrolidinyl group, anisoindolinyl group, or a piperazinyl group which is optionallysubstituted with a phenyl group. In another embodiment of Formula 1B,one or both R₂ groups is a sulfonyl group substituted with an —R_(b)group. In a further embodiment, one R₂ is a sulfonyl group substitutedwith an —R_(b) group and the other R₂ group is a hydrogen.

In another embodiment of Formula 1B, at least one R₂ group can be anaromatic group. In an embodiment of Formula 1B, one R₂ group is ahydrogen and the other R₂ group is a pyrimidinyl group or a pyridylgroup. In yet another embodiment of compounds of Formula 1B, one R₂group is a hydrogen and the other R₂ group is an —R_(b) group. Inaddition, compounds of Formula 1B include compounds where one R₂ is ahydrogen and the other is an R_(b) group, wherein the R_(b) group is aC₆ aryl group optionally substituted with one or more substituentsindependently selected from: hydroxy, halogen, C₁-C₄ alkyl, C₁-C₄haloalkyl, C₁-C₄ alkoxy, or an amino group which is optionallysubstituted with one or more independently selected C₁-C₄ alkyl groups.In another embodiment, compounds of Formula 1B include compounds whereone R₂ is a hydrogen and the other is an R_(b) group, wherein the R_(b)group is a phenyl group optionally substituted with one or moresubstituents independently selected from: hydroxy, halogen, C₁-C₄ alkyl,C₁-C₄ haloalkyl, C₁-C₄ alkoxy, or an amino group which is optionallysubstituted with one or more independently selected C₁-C₄ alkyl groups.In an embodiment of Formula 1B, X may be selected from the groupconsisting of fluorine, chlorine, bromine and iodine. In anotherembodiment of Formula 1B, X may be fluorine.

A variety of embodiments of compounds of Formula 1C are consideredwithin the scope of this invention. By way of non-limiting example, inan embodiment, three R_(c) groups of Formula 1C are hydrogen. In anotherembodiment, one R_(c) group is hydrogen and the other two R_(c) groupsare methyl. In yet another embodiment, two R_(c) groups are hydrogen andthe remaining R_(c) group is a methyl or an ethyl group. In anembodiment of Formula 1C, X may be selected from the group consisting offluorine, chlorine, bromine and iodine. In another embodiment of Formula1C, X may be fluorine.

As recognized by one of skill in the art, certain compounds of theinvention may include at least one chiral center, and as such may existas racemic mixtures or as enantiomerically pure compositions. As usedherein, “enantiomerically pure” refers to compositions consistingsubstantially of a single isomer, preferably consisting of 90%, 92%,95%, 98%, 99%, or 100% of a single isomer.

As used herein, the term “alkyl” generally refers to saturatedhydrocarbonyl radicals of straight, branched or cyclic, configurationincluding methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclohexyl, n-heptyl, octyl,n-octyl, and the like. In some embodiments, alkyl substituents may be C₁to C₈, C₁ to C₆, or C₁ to C₄ alkyl groups. In certain embodiments, thealkyl group may be optionally substituted with one or more halogen oralkoxy groups. For instance, the alkyl group may be a haloalkyl,including monohaloalkyl, dihaloalkyl, and trihaloalkyl.

As used herein, “alkylene” generally refers to linear, branched orcyclic alkene radicals having one or more carbon-carbon double bonds,such as C₂ to C₆ alkylene groups including 3-propenyl.

As used herein, “aryl” refers to a carbocyclic aromatic ring structure.Included in the scope of aryl groups are aromatic rings having from fiveto twenty carbon atoms. Aryl ring structures include compounds havingone or more ring structures, such as mono-, bi-, or tricyclic compounds.Non-limiting examples of aryl groups include phenyl, tolyl, anthracenyl,fluorenyl, indenyl, azulenyl, phenanthrenyl (i.e., phenanthrene), andnapthyl (i.e., napthalene) ring structures. In certain embodiments, thearyl group may be optionally substituted.

As used herein, “heterocycle” refers to cyclic ring structures in whichone or more atoms in the ring, the heteroatom(s), is an element otherthan carbon. Heteroatoms are typically O, S or N atoms. Included withinthe scope of heterocycle, and independently selectable, are O, N, and Sheterocycle ring structures. The ring structure may include compoundshaving one or more ring structures, such as mono-, bi-, or tricycliccompounds, and may be aromatic, i.e., the ring structure may be aheteroaryl. Example of heterocyclo groups include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl or tetrahydrothiopyranyl and the like. In certainembodiments, the heterocycle may optionally be substituted.

As used herein, “heteroaryl” refers to cyclic aromatic ring structuresin which one or more atoms in the ring, the heteroatom(s), is an elementother than carbon. Heteroatoms are typically O, S or N atoms. Includedwithin the scope of heteroaryl, and independently selectable, are O, N,and S heteroaryl ring structures. The ring structure may includecompounds having one or more ring structures, such as mono-, bi-, ortricyclic compounds. In some embodiments, the heteroaryl groups may beselected from heteroaryl groups that contain two or more heteroatoms,three or more heteroatoms, or four or more heteroatoms. Heteroaryl ringstructures may be selected from those that contain five or more atoms,six or more atoms, or eight or more atoms. In a preferred embodiment, aheteroaryl includes five to ten atoms. Examples of heteroaryl ringstructures include: acridine, benzimidazole, benzoxazole, benzodioxole,benzofuran, diazine, 1,2-diazine, 1,2-diazole, 1,4-diazanaphthalene,furan, furazan, imidazole, indole, isoxazole, isoquinoline, isothiazole,oxazole, purine, pyridazine, pyrazole, pyridine, pyrazine, pyrimidine,pyrrole, quinoline, quinoxaline, thiazole, thiophene, 1,3,5-triazine,1,2,3-triazine, tetrazole and quinazoline.

As used herein, “alkoxy” generally refers to a group with the structure—O—R. In certain embodiments, R may be an alkyl group, such as a C₁ toC₈, C₁ to C₆ alkyl group, or C₁ to C₄ alkyl group. In certainembodiments, the R group of the alkoxy may optionally be substitutedwith at least one halogen. For example, the R group of the alkoxy may bea haloalkyl, i.e., haloalkoxy.

Halogen substituents may be independently selected from the halogenssuch as fluorine, chlorine, bromine, iodine, and astatine.

For the purposes of this invention, where one or more functionalities orsubstituents are incorporated into a compound of the invention,including preferred embodiments, each functionality or substituentappearing at any location within the disclosed compounds may beindependently selected, and as appropriate, independently substituted.Further, where a more generic substituent is set forth for any positionin the molecules of the present invention, it is understood that thegeneric substituent may be replaced with more specific substituents, andthe resulting molecules are within the scope of the molecules of thepresent invention.

With reference to Formula 1, X is preferably fluorine and is preferablyin an ortho position. Preferred R₁ groups include those shown in thetable below.

Preferred R₁ Groups

Preferred R₂ groups include those shown in the table below,

Preferred R₂ Groups

Also with reference to Formula 1, preferred R groups include those shownin the table below.

Preferred R Groups

in a preferred embodiment, compounds of Formula 1 include the compoundsof Formula 1-A:

With reference to Formula 1-A, R₁ is preferably a C₁-C₈ alkyl groupoptionally substituted with one or more independently selected R_(a)groups. Further, X is preferably fluorine and is preferably located inan ortho position.

In another preferred embodiment, compounds of Formula 1 include thecompounds of Formula 1-B:

With reference to Formula 1-B, at least one R₂ group is preferably ahydrogen. Further, X is preferably fluorine and is preferably located inan ortho position.

In yet another preferred embodiment, compounds of Formula 1 include thecompounds of Formula 1-C.

With reference to Formula 1-C, R_(c) is independently selected fromhydrogen, a halogen, a methyl, or an ethyl. At least one R_(c) group ispreferably a fluorine. Further, X is preferably fluorine and ispreferably located in an ortho position.

Preferred compounds of the invention include the following.

Compounds

The above compounds are listed only to provide examples that may be usedin the methods of the invention. Based upon the instant disclosure, theskilled artisan would recognize other compounds intended to be includedwithin the scope of the presently claimed invention that would be usefulin the methods recited herein.

B. Preparation of Compounds of the Invention

Compounds of the invention may be produced in any manner known in theart. By way of example, compounds of the invention may be preparedaccording to the following general schemes with reference to theindividual R group structures. For example, triazine compounds ofFormulas 1-A, 1-B, and 1-C may be prepared in the manner shown in thefollowing Scheme A.

In certain preferred embodiments, compounds of the invention may beresolved to enantiomerically pure compositions or synthesized asenantiomerically pure compositions using any method known in art. By wayof example, compounds of the invention may be resolved by directcrystallization of enantiomer mixtures, by diastereomer salt formationof enantiomers, by the formation and separation of diasteriomers or byenzymatic resolution of a racemic mixture.

These and other reaction methodologies may be useful in preparing thecompounds of the invention, as recognized by one of skill in the art.Various modifications to the above schemes and procedures will beapparent to one of skill in the art, and the invention is not limitedspecifically by the method of preparing the compounds of the invention.

C. Methods of the Invention

In another aspect of the invention, methods are provided for thesuppression of premature translation termination, which may beassociated with a nonsense mutation, and for the prevention or treatmentof diseases. In a preferred embodiment, such diseases are associatedwith mutations of mRNA, especially nonsense mutations. Exemplarydiseases include, but are not limited to, cancer, lysosomal storagedisorders, the muscular dystrophies, cystic fibrosis, hemophilia,epidermolysis bullosa, and classical late infantile neuronal ceroidlipofuscinosis. In an embodiment, methods for treating cancer, lysosomalstorage disorders, the muscular dystrophies, cystic fibrosis,hemophilia, epidermolysis bullosa, and classical late infantile neuronalceroid lipofuscinosis are provided comprising administering atherapeutically effective amount of at least one compound of theinvention to a subject in need thereof. In another embodiment, methodsfor treating cancer, lysosomal storage disorders, a muscular dystrophy,cystic fibrosis, hemophilia, or classical late infantile neuronal ceroidlipofuscinosis are provided comprising administering a therapeuticallyeffective amount of at least one compound of the invention to a subjectin need thereof.

In one embodiment, the present invention is directed to methods forincreasing the expression of one or more specific, functional proteins.Any compound of the invention can be used to specifically increaseexpression of functional protein. In another embodiment, a specificincrease in expression of functional protein occurs when prematuretranslation termination is suppressed by administering a therapeuticallyeffective amount of at least one compound of the invention to a subjectin need thereof. In a preferred embodiment premature translationtermination is associated with a nonsense mutation in mRNA. In anotherembodiment, a specific increase in expression of functional proteinoccurs when mRNA decay is reduced in a patient. In a preferredembodiment, the abnormality in a patient is caused by mutation-mediatedmRNA decay. In a particularly preferred embodiment, mutation-mediatedmRNA decay is the result of a nonsense mutation. The methods of thepresent invention are not limited by any particular theory.

The invention encompasses methods of treating and preventing diseases ordisorders ameliorated by the suppression of premature translationtermination, nonsense-mediated mRNA decay, or premature translationtermination and nonsense-mediated mRNA decay in a patient which compriseadministering to a patient in need of such treatment or prevention atherapeutically effective amount of one or more compounds of theinvention.

In an embodiment, the present invention encompasses the treatment orprevention of any disease that is associated with a gene exhibitingpremature translation termination, nonsense-mediated mRNA decay, orpremature translation termination and nonsense-mediated mRNA decay. Inone embodiment, the disease is due, in part, to a lack of or a reducedexpression of the gene resulting from a premature stop codon. Specificexamples of genes which may exhibit premature translation terminationand/or nonsense-mediated mRNA decay and diseases associated withpremature translation termination and/or nonsense-mediated mRNA decayare found in U.S. Provisional Patent Application No. 60/390,747, titled:Methods For Identifying Small Molecules That Modulate PrematureTranslation Termination And Nonsense Mediated mRNA Decay, filed Jun. 21,2002, and International Application PCT/US03/19760, filed Jun. 23, 2003,both of which are incorporated herein by reference in their entireties.

Diseases ameliorated by the suppression of premature translationtermination, nonsense-mediated mRNA decay, or premature translationtermination and nonsense-mediated mRNA decay include, but are notlimited to: genetic diseases, somatic diseases, cancers, autoimmunediseases, blood diseases, collagen diseases, diabetes, neurodegenerativediseases, proliferative diseases, cardiovascular diseases, pulmonarydiseases, inflammatory diseases, or central nervous system diseases.

In one embodiment, diseases to be treated or prevented by administeringto a patient in need thereof an effective amount of a compound of theinvention include, but are not limited to, amyloidosis, hemophilia,Alzheimer's disease, Tay Sachs disease, Niemann Pick disease,atherosclerosis, giantism, dwarfism, hypothyroidism, hyperthyroidism,aging, obesity, Parkinson's disease, cystic fibrosis, musculardystrophy, heart disease, kidney stones, ataxia-telangiectasia, familialhypercholesterolemia, retinitis pigmentosa, Duchenne muscular dystrophy,epidermolysis bullosa and Marfan syndrome. In one embodiment, thediseases are associated with a nonsense mutation.

In one embodiment, the compounds of the invention are useful fortreating or preventing an autoimmune disease. In one embodiment, theautoimmune disease is associated with a nonsense mutation. In apreferred embodiment, the autoimmune disease is rheumatoid arthritis orgraft versus host disease.

In another embodiment, the compounds of the invention are useful fortreating or preventing a blood disease. In one embodiment, the blooddisease is associated with a nonsense mutation. In a preferredembodiment, the blood disease is hemophilia, Von Willebrand disease,β-thalassemia or kidney stones.

In another embodiment, the compounds of the invention are useful fortreating or preventing a collagen disease. In one embodiment, thecollagen disease is associated with a nonsense mutation. In a preferredembodiment, the collagen disease is osteogenesis imperfecta orcirrhosis.

In another embodiment, the compounds of the invention are useful fortreating or preventing diabetes. In one embodiment, the diabetes isassociated with a nonsense mutation.

In another embodiment, the compounds of the invention are useful fortreating or preventing an inflammatory disease. In one embodiment, theinflammatory disease is associated with a nonsense mutation. In apreferred embodiment, the inflammatory disease is arthritis, rheumatoidarthritis or osteoarthritis.

In another embodiment, the compounds of the invention are useful fortreating or preventing a central nervous system disease. In oneembodiment, the central nervous system disease is associated with anonsense mutation. In one embodiment, the central nervous system diseaseis a neurodegenerative disease. In a preferred embodiment, the centralnervous system disease is multiple sclerosis, muscular dystrophy,Duchenne muscular dystrophy. Alzheimer's disease, Tay Sachs disease,Niemann Pick disease, late infantile neuronal ceroid lipofuscinosis(LINCL) or Parkinson's disease.

In another preferred embodiment, the compounds of the invention areuseful for treating or preventing cancer, particularly in humans. In apreferred embodiment, the cancer is of the head and neck, eye, skin,mouth, throat, esophagus, chest, bone, blood, lung, colon, sigmoid,rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas,brain, intestine, heart or adrenals. In one embodiment, the cancer is asolid tumor. In one embodiment, the cancer is associated with a nonsensemutation. In another embodiment, the cancer is associated with a geneticnonsense mutation. In another embodiment, the cancer is associated witha somatic mutation. Without being limited by any theory, the use of thecompounds of the invention against cancer may relate to its actionagainst mutations of the p53 gene.

In one embodiment, the cancer is not a blood cancer. In anotherembodiment, the cancer is not leukemia. In another embodiment, thecancer is not multiple myeloma. In another embodiment, the cancer is notprostate cancer.

In another preferred embodiment, the compounds of the invention areuseful for treating or preventing cancer associated with a mutation oftumor suppressor gene. Such genes include, but are not limited to, PTEN,BRCA1, BRCA2, Rb, and the p53 gene. In one embodiment, the mutation is agenetic mutation. In another embodiment, the mutation is a somaticmutation. The methods of the invention are particularly useful fortreating or preventing a cancer associated with a nonsense mutation inthe in a tumor suppressor gene. In a preferred embodiment, the methodsof the invention are particularly useful for treating or preventing acancer associated with a p53 gene due to the role of p53 in apoptosis.Without being limited by theory, it is thought that apoptosis can beinduced by contacting a cell with an effective amount of a compound ofthe invention resulting in suppression of the nonsense mutation, which,in turn, allows the production of full-length p53 to occur. Nonsensemutations have been identified in the p53 gene and have been implicatedin cancer. Several nonsense mutations in the p53 gene have beenidentified (see, e.g., Masuda et al., 2000, Tokai J Exp Clin Med.25(2):69-77; Oh et al., 2000, Mol Cells 10(3):275-80; Li et al., 2000,Lab Invest. 80(4):493-9; Yang et al., 1999. Zhonghua Zhong Liu Za Zhi21(2):114-8; Finkelstein at al., 1998, Mol Diagn. 3(1):37-41; Kajiyamaet al., 1998, Dis Esophagus. 11(4):279-83; Kawamura et al., 1999, LeukRes. 23(2):115-26; Radig et al., 1998, Hum Pathol. 29(11):1310-6;Schuyer et al., 1998, Int J Cancer 76(3):299-303; Wang-Gohrke et al.,1998, Oncol Rep. 5(1):65-8; Fulop et al., 1998, J Reprod Med.43(2):119-27; Ninomiya et al., 1997, J Dermatol Sci. 14(3):173-8; Hsiehet al., 1996, Cancer Lett. 100(1-2):107-13; Rall et al., 1996, Pancreas.12(1):10-7; Fukutomi et al., 1995, Nippon Rinsho. 53(11):2764-8;Frebourg, et al., 1995, Am J Hum Genet. 56(3):608-15; Dove et al., 1995,Cancer Surv. 25:335-55; Adamson et al., 1995, Br J Haematol. 89(1):61-6;Grayson et al., 1994, Am J Pediatr Hematol Oncol. 16(4):341-7; Lepelleyet al., 1994, Leukemia, 8(8):1342-9; McIntyre et al., 1994, J ClinOncol. 12(5):925-30; Horio et al., 1994, Oncogene, 9(4):1231-5; Nakamuraet al., 1992, Jpn J Cancer Res. 83(12):1293-8; Davidoff et al., 1992,Oncogene. 7(1):127-33; and Ishioka et al., 1991, Biochem Biophys ResCommun. 177(3):901-6; the disclosures of which are hereby incorporatedby reference herein in their entireties). Any disease associated with ap53 gene encoding a premature translation codon including, but notlimited to, the nonsense mutations described in the references citedabove, may be treated or prevented by compounds of the invention.

In other embodiments, diseases to be treated or prevented byadministering to a patient in need thereof an effective amount of acompound of the invention include, but are not limited to, solid tumorssuch as sarcoma, carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, retinoblastoma, a blood-born tumor or multiplemyeloma.

In another embodiment, diseases to be treated or prevented byadministering to a patient in need thereof an effective amount of acompound of the invention include, but are not limited to, a blood-borntumor such as acute lymphoblastic leukemia, acute lymphoblastic B-cellleukemia, acute lymphoblastic T-cell leukemia, acute myeloblasticleukemia, acute promyelocytic leukemia, acute monoblastic leukemia,acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acutemyelomonocytic leukemia, acute nonlymphocyctic leukemia, acuteundifferentiated leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, hairy cell leukemia, or multiple myeloma. Seee.g., Harrison's Principles of Internal Medicine, Eugene Braunwald etal., eds., pp. 491-762 (15th ed. 2001).

In yet another embodiment, the invention encompasses the treatment of ahuman afflicted with a solid tumor or a blood tumor.

In a preferred embodiment, the invention encompasses a method oftreating or preventing a disease ameliorated by modulation of prematuretranslation termination, nonsense-mediated mRNA decay, or prematuretranslation termination and nonsense-mediated mRNA decay, orameliorating one or more symptoms associated therewith comprisingcontacting a cell with a therapeutically effective amount of a compoundof the invention. Cells encompassed by the present methods includeanimal cells, mammalian cells, bacterial cells, and virally infectedcells. In one embodiment, the nonsense mutation is a genetic mutation(i.e., the nonsense codon was present in the progenitor DNA). In anotherembodiment, the nonsense mutation is a somatic mutation (i.e., thenonsense codon arose spontaneously or from mutagenesis).

In certain embodiments, a compound of the invention is administered to asubject, including but not limited to a plant, reptile, avian, amphibianor preferably a mammal, more preferably a human, as a preventativemeasure against a disease associated with premature translationtermination, nonsense-mediated mRNA decay, or premature translationtermination and nonsense-mediated mRNA decay.

In a preferred embodiment, it is first determined that the patient issuffering from a disease associated with premature translationtermination and/or nonsense-mediated mRNA decay. In another embodiment,the patient has undergone a screening process to determine the presenceof a nonsense mutation comprising the steps of screening a subject, orcells extracted therefrom, by an acceptable nonsense mutation screeningassay. In a preferred embodiment, the DNA of the patient can besequenced or subjected to Southern Blot, polymerase chain reaction(PCR), use of the Short Tandem Repeat (STR), or polymorphic lengthrestriction fragments (RFLP) analysis to determine if a nonsensemutation is present in the DNA of the patient. In one embodiment, it isdetermined whether the nonsense mutation is a genetic mutation or asomatic mutation by comparison of progenitor DNA. Alternatively, it canbe determined if altered levels of the protein with the nonsensemutation are expressed in the patient by Western blot or otherimmunoassays. In another embodiment, the patient is an unborn child whohas undergone screening in utero for the presence of a nonsensemutation. Administration of a compound of the invention can occur eitherbefore or after birth. In a related embodiment, the therapy ispersonalized in that the patient is screened for a nonsense mutationscreening assay and treated by the administration of one or morecompounds of the invention; particularly, the patient may be treatedwith a compound particularly suited for the mutations in question; e.g.,depending upon the disease type, cell type, and the gene in question.Such methods are well known to one of skill in the art.

In another embodiment, the cells (e.g., animal cells, mammalian cells,bacterial cells, plant cells and virally infected cells) are screenedfor premature translation termination and/or nonsense-mediated mRNAdecay with a method such as that described above (i.e., the DNA of thecell can be sequenced or subjected to Southern Blot, polymerase chainreaction (PCR), use of the Short Tandem Repeat (STR), or polymorphiclength restriction fragments (RFLP) analysis to determine if a nonsensemutation is present in the DNA of the cell; the RNA of the cell can besubjected to quantitative real time PCR to determine transcriptabundance).

Specific methods of the invention further comprise the administration ofan additional therapeutic agent (i.e., a therapeutic agent other than acompound of the invention), in certain embodiments of the presentinvention, the compounds of the invention can be used in combinationwith at least one other therapeutic agent. Therapeutic agents include,but are not limited to: non-opioid analgesics; non-steroidanti-inflammatory agents; steroids, antimimetics; β-adrenergic blockers;anticonvulsants; antidepressants; Ca²⁺-channel blockers; anticanceragent(s); antibiotics; and mixtures thereof.

In certain embodiments, the compounds of the invention can beadministered or formulated in combination with anticancer agents.Suitable anticancer agents include, but are not limited to: alkylatingagents; nitrogen mustards; folate antagonists; purine antagonists;pyrimidine antagonists; spindle poisons; topoisomerase inhibitors;apoptosis inducing agents; angiogenesis inhibitors; podophyllotoxins;nitrosoureas; cisplatin; carboplatin; interferon; asparginase;tamoxifen; leuprolide; flutamide; megestrol; mitomycin; bleomycin;doxorubicin; irinotecan; and taxol.

In certain embodiments, the compounds of the invention can beadministered or formulated in combination with antibiotics. In certainembodiments, the antibiotic is an aminoglycoside (e.g., tobramycin), acephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil,cefaclor, cefixime or cefadroxil), a clarithromycin (e.g.,clarithromycin), a macrolide (e.g., erythromycin), a penicillin (e.g.,penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin ornorfloxacin). In a preferred embodiment, the antibiotic is activeagainst Pseudomonas aeruginosa.

Without intending to be limited by theory, it is believed that themethods of the present invention act through a combination of mechanismsthat suppress nonsense mutations. In preferred embodiments, the methodsof the invention comprise administering a therapeutically effectiveamount of at least one compound of the invention, e.g., a compound ofFormula 1. Relative activity of the compounds of the invention may bedetermined by any method known in the art, including for example, theassay described in Example 2 herein.

Compounds of the invention can be characterized with an in vitroluciferase nonsense suppression assay. Luciferase assays are included inthe methods of the present invention. Luciferase can be used as afunctional reporter gene assay (light is only produced if the protein isfunctional), and luciferase is extremely sensitive (light intensity isproportional to luciferase concentration in the nM range). In oneembodiment, an assay of the present invention is a cell-based luciferasereporter assay. In a preferred cell-based luciferase reporter assay, aluciferase reporter construct containing a premature termination codon(UGA, UAA, or UAG) is stably transfected in 293 Human Embryonic Kidneycells.

In another assay of the present invention, a preferred assay is abiochemical assay consisting of rabbit reticulocyte lysate and anonsense-containing luciferase reporter mRNA. In another assay of thepresent invention, the assay is a biochemical assay consisting ofprepared and optimized cell extract (Lie & Macdonald, 1999, Development126(22):4989-4996 and Lie & Macdonald, 2000, Biochem. Biophys. Res.Commun. 270(2):473-481. In the biochemical assay, mRNA containing apremature termination codon (UGA, UAA, or UAG) is used as a reporter inan in vitro translation reaction using rabbit reticulocyte lysatesupplemented with tRNA, hemin, creatine kinase, amino acids, KOAc,Mg(OAc)₂, and creatine phosphate. Translation of the mRNA is initiatedwithin a virus derived leader sequence, which significantly reduces thecost of the assay because capped RNA is not required. Synthetic mRNA isprepared in vitro using the T7 promoter and the MegaScript in vitrotranscription kit (Ambion, Inc.; Austin, Tex.). In assays of the presentinvention, addition of gentamicin, an aminoglycoside known to allowreadthrough of premature termination codons, results in increasedluciferase activity and can be used as an internal standard. Assays ofthe present invention can be used in high-throughput screens. Hundredsof thousands of compounds can be screened in cell-based and biochemicalassays of the present invention. In a preferred aspect, a functionalcell-based assay similar to the one described is provided.

Compounds of the present invention include compounds capable ofincreasing specific, functional protein expression from mRNA moleculescomprising premature termination codons. In one embodiment, compounds ofthe present invention can preferentially suppress premature translationtermination. For example, a compound of the present invention can becapable of suppressing a nonsense mutation if the mutation results inUAA, but not capable of suppressing a nonsense mutation if the mutationresults in UAG. Another non-limiting example occurs when a compound ofthe present invention is capable of suppressing a nonsense mutation ifthe mutation results in UAA and is followed, in-frame by a cytosine atthe +1 position, but the same compound of the invention is not capableof suppressing a nonsense mutation if the mutation results in UAA and isfollowed, in-frame by an adenine at the +1 position.

A stable cell line harboring the UGA nonsense-containing luciferase genecan be treated with a test compound. In this aspect, cells can be grownin standard medium supplemented with 1% penicillin-streptomycin (P/S)and 10% fetal bovine serum (FBS) to 70% confluency and split 1:1 the daybefore treatment. The next day, cells are trypsinized and 40,000 cellsare added to each well of a 96-well tissue culture dish. Serialdilutions of each compound are prepared to generate a six-point doseresponse curve spanning 2 logs (30 μM to 0.3 μM). The finalconcentration of the DMSO solvent remains constant at 1% in each well.Cells treated with 1% DMSO serve as the background standard, and cellstreated with gentamicin serve as a positive control.

To address the effects of the nonsense-suppressing compounds on mRNAsaltered in specific inherited diseases, a bronchial epithelial cell lineharboring a nonsense codon at amino acid 1282 (W1282X) can be treatedwith a compound of the invention and CFTR function is monitored as acAMP-activated chloride channel using the SPQ assay (Yang et al., Hum.Mol. Genet. 2(8):1253-1261 (1993) and Howard et al., Nat. Med.2(4):467-4691996)). The increase in SPQ fluorescence in cells treatedwith a compound of the invention is compared to those treated with cAMPand untreated cells. An increase in SPQ fluorescence in cells isconsistent with stimulation of CFTR-mediated halide efflux and anincrease in readthrough of the nonsense codon. Full-length CFTRexpression from this nonsense-containing allele following treatment witha compound of the invention demonstrates that cystic fibrosis cell linesincrease chloride channel activity when treated with a compound of theinvention.

D. Metabolites of the Compounds of the Invention

Also falling within the scope of the present invention are the in vivometabolic products of the compounds described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,esterification and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising contacting a compound of this inventionwith a mammalian tissue or a mammal for a period of time sufficient toyield a metabolic product thereof. Such products typically areidentified by preparing a radio-labeled (e.g. ¹⁴C or ³H) compound of theinvention, administering it in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to a mammal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to about 30 hours), and isolating its conversionproducts from urine, blood or other biological samples. These productsare easily isolated since they are labeled (others are isolated by theuse of antibodies capable of binding epitopes surviving in themetabolite). The metabolite structures are determined in conventionalfashion, e.g., by MS or NMR analysis. In general, analysis ofmetabolites may be done in the same way as conventional drug metabolismstudies well-known to those skilled in the art. The conversion products,so long as they are not otherwise found in vivo, are useful indiagnostic assays for therapeutic dosing of the compounds of theinvention even if they possess no biological activity of their own.

E. Pharmaceutical Compositions of the Invention

While it is possible for the compounds of the present invention to beadministered neat, it may be preferable to formulate the compounds aspharmaceutical compositions. As such, in yet another aspect of theinvention, pharmaceutical compositions useful in the methods of theinvention are provided. The pharmaceutical compositions of the presentinvention may be formulated with pharmaceutically acceptable excipientssuch as carriers, solvents, stabilizers, adjuvants, diluents, etc.,depending upon the particular mode of administration and dosage form.The pharmaceutical compositions should generally be formulated toachieve a physiologically compatible pH, and may range from a pH ofabout 3 to a pH of about 11, preferably about pH 3 to about pH 7, orabout pH 4 to about pH 7, depending on the formulation and route ofadministration. In an alternative embodiment, it may be preferred thatthe pH is adjusted to a range from about pH 5 to about pH 8.

More particularly, the pharmaceutical compositions of the inventioncomprise a therapeutically or prophylactically effective amount of atleast one compound of the present invention, together with one or morepharmaceutically acceptable excipients. Optionally, the pharmaceuticalcompositions of the invention may comprise a combination of compounds ofthe present invention, or may include a second active ingredient usefulin the treatment of cancer, diabetic retinopathy, or exudative maculardegeneration.

Formulations of the present invention, e.g., for parenteral or oraladministration, are most typically solids, liquid solutions, emulsionsor suspensions, while inhalable formulations for pulmonaryadministration are generally liquids or powders, with powderformulations being generally preferred. A preferred pharmaceuticalcomposition of the invention may also be formulated as a lyophilizedsolid that is reconstituted with a physiologically compatible solventprior to administration. Alternative pharmaceutical compositions of theinvention may be formulated as syrups, creams, ointments, tablets, andthe like.

The pharmaceutical compositions of the invention can be administered tothe subject via any drug delivery route known in the art. Specificexemplary administration routes include oral, ocular, rectal, buccal,topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous(bolus and infusion), intracerebral, transdermal, and pulmonary.

The term “pharmaceutically acceptable excipient” refers to an excipientfor administration of a pharmaceutical agent, such as the compounds ofthe present invention. The term refers to any pharmaceutical excipientthat may be administered without undue toxicity. Pharmaceuticallyacceptable excipients are determined in part by the particularcomposition being administered, as well as by the particular method usedto administer the composition. Accordingly, there exists a wide varietyof suitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed.,Mack Publishing Co, 1990).

Suitable excipients may be carrier molecules that include large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Other exemplary excipients includeantioxidants such as ascorbic acid; chelating agents such as EDTA;carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethylcellulose, stearic acid; liquids such as oils, water,saline, glycerol and ethanol; wetting or emulsifying agents; pHbuffering substances; and the like. Liposomes are also included asexemplary pharmaceutically acceptable excipients.

The pharmaceutical compositions of the invention may be formulated inany form suitable for the intended method of administration. Whenintended for oral use for example, tablets, troches, lozenges, aqueousor oil suspensions, non-aqueous solutions, dispersible powders orgranules (including micronized particles or nanoparticles), emulsions,hard or soft capsules, syrups or elixirs may be prepared. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions, and suchcompositions may contain one or more agents including sweetening agents,flavoring agents, coloring agents and preserving agents, in order toprovide a palatable preparation.

Pharmaceutically acceptable excipients particularly suitable for use inconjunction with tablets include, for example, inert diluents, such ascelluloses, calcium or sodium carbonate, lactose, calcium or sodiumphosphate; disintegrating agents, such as croscarmellose sodium,cross-linked povidone, maize starch, or alginic acid; binding agents,such as povidone, starch, gelatin or acacia; and lubricating agents,such as magnesium stearate, stearic acid or talc. Tablets may beuncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample celluloses, lactose, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with non-aqueousor oil medium, such as glycerin, propylene glycol, polyethylene glycol,peanut oil, liquid paraffin or olive oil.

In another embodiment, pharmaceutical compositions of the invention maybe formulated as suspensions comprising a compound of the presentinvention in admixture with at least one pharmaceutically acceptableexcipient suitable for the manufacture of a suspension. In yet anotherembodiment, pharmaceutical compositions of the invention may beformulated as dispersible powders and granules suitable for preparationof a suspension by the addition of suitable excipients.

Excipients suitable for use in connection with suspensions includesuspending agents, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methylcelluose, sodium alginate,polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or wettingagents such as a naturally occurring phosphatide (e.g., lecithin), acondensation product of an alkylene oxide with a fatty acid (e.g.,polyoxyethylene stearate), a condensation product of ethylene oxide witha long chain aliphatic alcohol (e.g., heptadecaethyleneoxycethanol), acondensation product of ethylene oxide with a partial ester derived froma fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitanmonooleate); and thickening agents, such as carbomer, beeswax, hardparaffin or cetyl alcohol. The suspensions may also contain one or morepreservatives such as acetic acid, methyl and/or n-propylp-hydroxy-benzoate; one or more coloring agents; one or more flavoringagents; and one or more sweetening agents such as sucrose or saccharin.

The pharmaceutical compositions of the invention may also be in the forof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of oils. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth;naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids; hexitol anhydrides, such assorbitan monooleate; and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents. Syrups andelixirs may be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such formulations may also contain a demulcent, apreservative, a flavoring or a coloring agent.

Additionally, the pharmaceutical compositions of the invention may be inthe form of a sterile injectable preparation, such as a sterileinjectable aqueous emulsion or oleaginous suspension. This emulsion orsuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, such as a solution in 1,2-propane diol.The sterile injectable preparation may also be prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid may likewise be used in the preparation of injectables.

Generally, the compounds of the present invention useful in the methodsof the present invention are substantially insoluble in water and aresparingly soluble in most pharmaceutically acceptable protic solventsand in vegetable oils. However, the compounds are generally soluble inmedium chain fatty acids (e.g., caprylic and capric acids) ortriglycerides and have high solubility in propylene glycol esters ofmedium chain fatty acids. Also contemplated in the invention arecompounds which have been modified by substitutions or additions ofchemical or biochemical moieties which make them more suitable fordelivery (e.g., increase solubility, bioactivity, palatability, decreaseadverse reactions, etc.), for example by esterification, glycosylation,PEGylation, etc.

In a preferred embodiment, the compounds of the present invention may beformulated for oral administration in a lipid-based formulation suitablefor low solubility compounds. Lipid-based formulations can generallyenhance the oral bioavailability of such compounds. As such, a preferredpharmaceutical composition of the invention comprises a therapeuticallyor prophylactically effective amount of a compound of the presentinvention, together with at least one pharmaceutically acceptableexcipient selected from the group consisting of: medium chain fattyacids or propylene glycol esters thereof (e.g., propylene glycol estersof edible fatty acids such as caprylic and capric fatty acids) andpharmaceutically acceptable surfactants such as polyoxyl 40 hydrogenatedcastor oil.

In an alternative preferred embodiment, cyclodextrins may be added asaqueous solubility enhancers. Preferred cyclodextrins includehydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosylderivatives of α-, β-, and γ-cyclodextrin. A particularly preferredcyclodextrin solubility enhancer is hydroxypropyl-β-cyclodextrin (HPBC),which may be added to any of the above-described compositions to furtherimprove the aqueous solubility characteristics of the compounds of thepresent invention. In one embodiment, the composition comprises 0.1% to20% hydroxypropyl-β-cyclodextrin, more preferably 1% to 15%hydroxypropyl-β-cyclodextrin, and even more preferably from 2.5% to 10%hydroxypropyl-β-cyclodextrin. The amount of solubility enhancer employedwill depend on the amount of the compound of the present invention inthe composition.

A therapeutically effective amount, as used herein, refers to an amountof a pharmaceutical composition of the invention to treat, ameliorate,or modulate an identified disease or condition, or to exhibit adetectable therapeutic or inhibitory effect. The effect can be detectedby, for example, assays of the present invention. The effect can also bethe prevention of a disease or condition where the disease or conditionis predicted for an individual or a high percentage of a population.

The precise effective amount for a subject will depend upon a variety offactors such as for example the subject's body weight, size, and health;the nature and extent of the condition; the therapeutic or combinationof therapeutics selected for administration, the protein half-life, themRNA half-life and the protein localization. Therapeutically effectiveamounts for a given subject can be determined by routine experimentationthat is within the skill and judgment of the clinician.

For any compound, the therapeutically effective amount car be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD50/ED50. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies may be used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include an ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

More specifically, the concentration-biological effect relationshipsobserved with regard to the compound(s) of the present inventionindicate an initial target plasma concentration ranging fromapproximately 5 μg/mL to approximately 100 μg/mL, preferably fromapproximately 10 μg/mL to approximately 50 μg/mL, more preferably fromapproximately 10 μg/mL to approximately 25 μg/mL. To achieve such plasmaconcentrations, the compounds of the invention may be administered atdoses that vary from 1 mg/kg to 150 mg/kg, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and is generally available topractitioners in the art. In general the dose will be in the range ofabout 1 mg/day to about 10 g/day, or about 0.1 g to about 3 g/day, orabout 0.3 g to about 3 g/day, or about 0.5 g to about 2 g/day, insingle, divided, or continuous doses for a patient weighing betweenabout 40 to about 100 kg (which dose may be adjusted for patients aboveor below this weight range, particularly children under 40 kg).

The magnitude of a prophylactic or therapeutic dose of a particularactive ingredient of the invention in the acute or chronic management ofa disease or condition will vary, however, with the nature and severityof the disease or condition, and the route by which the activeingredient is administered. The dose, and perhaps the dose frequency,will also vary according to the age, body weight, and response of theindividual patient. Suitable dosing regimens can be readily selected bythose skilled in the art with due consideration of such factors. Ingeneral, the recommended daily dose range for the conditions describedherein lie within the range of from about 1 mg/kg to about 150 mg/kg perday. In one embodiment, the compound of the invention is given as asingle once-a-day dose. In another embodiment, the compound of theinvention is given as divided doses throughout a day. More specifically,the daily dose is administered in a single dose or in equally divideddoses. Preferably, a daily dose range should be from about 5 mg/kg toabout 100 mg/kg per day, more preferably, between about 10 mg/kg andabout 90 mg/kg per day, even more preferably 20 mg/kg to 60 mg/kg perday. In managing a patient, the therapy should be initiated at a lowerdose, perhaps about 200 mg to about 300 mg, and increased if necessaryup to about 600 mg to about 4000 mg per day as either a single dose ordivided doses, depending on the patient's global response. It may benecessary to use dosages of the active ingredient outside the rangesdisclosed herein in some cases, as will be apparent to those of ordinaryskill in the art. Furthermore, it is noted that the clinician ortreating physician will know how and when to interrupt, adjust, orterminate therapy in conjunction with individual patient response.

Different therapeutically effective amounts may be applicable fordifferent diseases and conditions, as will be readily known by those ofordinary skill in the art. Similarly, amounts sufficient to treat orprevent such diseases, but insufficient to cause, or sufficient toreduce, adverse effects associated with conventional therapies are alsoencompassed by the above described dosage amounts and dose frequencyschedules.

As stated before. the exact dosage will be determined by thepractitioner, in light of factors related to the subject that requirestreatment. Dosage and administration are adjusted to provide sufficientlevels of the active agent(s) or to maintain the desired effect. Factorswhich may be taken into account include the severity of the diseasestate, general health of the subject, age, weight, and gender of thesubject, diet, time, protein of interest half-life, RNA of interesthalf-life, frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

F. Combination Therapy

It is also possible to combine one or more compounds of the presentinvention with one or more other active ingredients useful in thetreatment of diseases associated with nonsense mutations of mRNA asdescribed herein, including compounds in a unitary dosage form, or inseparate dosage forms intended for simultaneous or sequentialadministration to a patient in need of treatment. When administeredsequentially, the combination may be administered in two or moreadministrations. In an alternative embodiment, it is possible toadminister one or more compounds of the present invention and one ormore additional active ingredients by different routes.

The skilled artisan will recognize that a variety of active ingredientsmay be administered in combination with the compounds of the presentinvention that may act to augment or synergistically enhance thenonsense mutation-suppressing activity of the compounds of theinvention.

According to the methods of the invention, the combination of activeingredients may be: (1) co-formulated and administered or deliveredsimultaneously in a combined formulation; (2) delivered by alternationor in parallel as separate formulations; or (3) by any other combinationtherapy regimen known in the art. When delivered in alternation therapy,the methods of the invention may comprise administering or deliveringthe active ingredients sequentially, e.g., in separate solution,emulsion, suspension, tablets, pills or capsules, or by differentinjections in separate syringes. In general, during alternation therapy,an effective dosage of each active ingredient is administeredsequentially, i.e., serially, whereas in simultaneous therapy, effectivedosages of two or more active ingredients are administered together.Various sequences of intermittent combination therapy may also be used.

G. Gene Therapy

The compounds of the present invention or other nonsense compounds canbe utilized in combination with gene therapy. In this embodiment, a genecan be introduced or provided to a mammal, preferably a human thatcontains a specified nonsense mutation in the desired gene. In apreferred aspect, the desired gene is selected from the group consistingof IGF1, EPO, p53, p19ARF, p21, PTEN, EI 24 and ApoAI. In order toobtain expression of the full-length polypeptide in a patient or mammal,the patient or mammal would be provided with an effective amount of acompound of the present invention or other nonsense suppression compoundwhen such polypeptide is desired.

There are typically two major approaches to getting nucleic acids thatcontain a nonsense mutation (optionally contained in a vector) into apatient's cells: in vivo and ex vivo. For in vivo delivery the nucleicacid is injected directly into the patient, usually at the sites wherethe polypeptide is required, i.e., the site of synthesis of thepolypeptide, if known, and the site (e.g. solid tumor) where biologicalactivity of the polypeptide is needed. For ex vivo treatment, thepatient's cells are removed, the nucleic acid is introduced into theisolated cells, and the modified cells are administered to the patienteither directly or, for example, encapsulated within porous membranesthat are implanted into the patient (see e.g., U.S. Pat. Nos. 4,892,538and 5,283,187). There are a variety of techniques available forintroducing nucleic acids into viable cells. The techniques varydepending upon whether the nucleic acid is transferred into culturedcells in vitro, or transferred in vivo in the cells of the intendedhost. Techniques suitable for the transfer of nucleic acid intomammalian cells in vitro include the use of liposomes, electroporation,microinjection, transduction, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. Transduction involves theassociation of a replication-defective, recombinant viral (preferablyretroviral) particle with a cellular receptor, followed by introductionof the nucleic acids contained by the particle into the cell. A commonlyused vector for ex vivo delivery of the gene is a retrovirus.

The currently preferred in vivo nucleic and transfer techniques includetransfection with viral or non-viral vectors (such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) andlipid-based systems (useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol; see, e.g., Tonkinson etal., Cancer Investigation, 14 (1): 54-65 (1996)). The most preferredvectors for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral vector such asa retroviral vector includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger. Inaddition, a viral vector such as a retroviral vector includes a nucleicacid sequence that, when transcribed with a gene encoding a polypeptide,is operably linked to the coding sequence and acts as a translationinitiation sequence. Such vector constructs also include a packagingsignal, long terminal repeats (LTRs) or portions thereof, and positiveand negative strand primer binding sites appropriate to the virus used(if these are not already present in the viral vector). In addition,such vector typically includes a signal sequence for secretion of thepolypeptide from a host cell in which it is placed. Preferably thesignal sequence for this purpose is a mammalian signal sequence, mostpreferably the native signal sequence for the polypeptide. Optionally,the vector construct may also include a signal that directspolyadenylation, as well as one or more restriction sites and atranslation termination sequences. By way of example, such vectors willtypically include a 5′ LTR, a tRNA binding site, a packaging signal, aorigin of second-strand DNA synthesis, and a 3′ LTR or a portionthereof. Other vectors can be used that are non-viral, such as cationiclipids, polylysine, and dendrimers.

In some situations, it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell-surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins that bind to a cell-surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins that undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262: 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87: 3410-3414 (1990). For a review of the currentlyknown gene marking and gene therapy protocols, see, Anderson et al.,Science 256: 808-813 (1992). See also WO 93/25673 and the referencescited therein.

Suitable gene therapy and methods for making retroviral particles andstructural proteins can be found in, e.g. U.S. Pat. Nos. 5,681,746;6,800,604 and 6,800,731.

To assist in understanding the present invention, the following Examplesare included. The experiments relating to this invention should not, ofcourse, be construed as specifically limiting the invention and suchvariations of the invention, now known or later developed, which wouldbe within the purview of one skilled in the art are considered to fallwithin the scope of the invention as described herein and hereinafterclaimed.

EXAMPLES

The present invention is described in more detail with reference to thefollowing non-limiting examples, which are offered to more fullyillustrate the invention, but are not to be construed as limiting thescope thereof. The examples illustrate the preparation of certaincompounds of the invention, and the testing of these compounds in vitroand/or in vivo. Those of skill in the art will understand that thetechniques described in these examples represent techniques described bythe inventors to function well in the practice of the invention, and assuch constitute preferred modes for the practice thereof. However, itshould be appreciated that those of skill in the art should in light ofthe present disclosure, appreciate that many changes can be made in thespecific methods that are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Preparation of Compounds of the Invention A. Compound 25:Morpholine-4-carboxylic acid3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoylamide

Compound 25 of the present invention may be prepared according to SchemeA as follows. A slurry of3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid (Compound A1,1.5.0 g; 52.8 mmol) and 100 μL of dimethylformamide catalyst in 250 mLof anhydrous dichloromethane is allowed to cool to 0° C. Oxalyl chloride(9.2 mL, 105.5 mmol, 2.0 eq.) is added to this slurry over 3 minutes.The reaction mixture is then allowed to warm to room temperature, and isstirred for 16 h. The resultant yellow solution is concentrated invacuo, azeotroped several times with anhydrous dichloromethane (2×50 mL)and dried under high vacuum for 2 hrs to give 15.5 g of3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoyl chloride (CompoundA2) as a white solid (97%): ¹H NMR (CDCl₃) δ 7.23-7.38 (m, 2H),7.58-7.71 (m, 2H), 8.22-8.29 (m, 2H), 8.50 (d, J=7.8 Hz, 1H), 8.91 (t,J=1.6 Hz, 1H).

Saturated NH₃/THF solution (130 ml) cooled to 0° C. via cannula slowlyover 10 min is then added to a suspension of3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-benzoyl chloride (CompoundA2, 8.0 g, 26.43 mmol) in anhydrous tetrahydrofuran (80 mL) cooled to−78′C. The mixture is stirred for 30 minutes, warmed to room temperatureand stirred for 16 h. The solvent is concentrated in vacuo and theresulting white residue is washed several times with water. The wetsolid is dried in a vacuum oven for two days to give 7.06 g (94%) of3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzamide (Compound A3) as awhite solid: mp 231-234° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 6.10 (br d,J=9.7 Hz, 2H) 7.28-7.39 (m, 2H), 7.64 (t, J=7.8 Hz, 2H), 8.07 (d, J=7.8Hz, 1H), 8.24 (t, J=7.5 Hz, 1H), 8.37 (d, J=7.5 Hz, 1H), 8.58 (t, J=1.8Hz, 1H); MS m/z 284.12, calcd for C₁₅H₁₀FN₃O₂ (MH⁺) 284.

Oxalyl chloride (2.5 ml, 28.2 mmol, 2.0 eq.) is then added dropwiseunder N₂ to a suspension of3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzamide (Compound A3, 4.0g, 14.1 mmol) in anhydrous dichloromethane (20 mL) cooled to 0° C. Themixture is slowly heated to reflux and stirred for an additional 6 h.The solvent is removed on a rotary evaporator. The residue is azeotropedwith anhydrous dichloromethane (20 mL) and dried under high vacuum fortwo hours to give 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoylisocyanate (Compound A4) as a tan solid 4.12 g (95%) which may be usedimmediately without further purification.

Morpholine (114 μL; 1.30 mmol, 2.0 eq.) is added to a solution of3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoyl isocyanate (CompoundA4, 200 mg, 0.65 mmol) in anhydrous dichloromethane (4.0 mL) undernitrogen, and the reaction mixture is heated to reflux for 18 h. Theresulting slurry is filtered and recrystallized from the minimum amountof dichloromethane in hexanes to afford 249 mg (97%) ofmorpholine-4-carboxylic acid3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoylamide (Compound 25)as a white powder: mp 190-194° C.; ¹H NMR (300 MHz, DMSO-d₅) δ 3.05 (t,5.1 Hz, 2H), 3.46 (d, 4.5 Hz, 2H), 3.61 (m, 2H), 3.76 (t, J=5.1 Hz, 2H),7.46-7.90 (m, 4H), 7.59 (d, J=8.1 Hz, 1H), 8.22-8.29 (m, 2H), 8.53 (d,J=1.5 Hz, 1H), 9.20 (br s, 1H); MS m/z 397.15, calcd for C₂₀H₁₇FN₄O₄(MH⁺) 397.

B. Compound 37:(±)-{3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]benzoyl}carbamic acid1,2,2-trimethylpropyl ester

3,3-dimethyl-2-butanol (306 μL; 2.43 mmol, 5.0 eq.) is added to asolution of 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoylisocyanate (Compound A4, 150 mg; 0.49 mmol) in anhydrous dichloromethane(4.0 mL) in 1-portion, and then the reaction mixture is heated to refluxfor 18 h. The crude residue is purified by silica gel chromatographyusing a 10 g SiO₂ column (eluted with 35% ethyl acetate/hexane) to give140 mg (70% yield) of a white powder (Compound 37): mp 170-173; ¹H NMR(300 MHz, DMSO-d₆) δ 0.92 (s, 9H), 1.17 (d, J=6.6 Hz, 3H), 4.60 (q,J=6.6 Hz, 1H), 7.46-7.85 (m, 4H), 8.07 (d, J=7.8 Hz, 1H), 8.23-8.30 (m,2H), 8.49 (s, 1H), 11.08 (s, 1H); MS m/z 412.13, calcd for C₂₂H₂₂FN₃O₄(MH⁺) 412.

C. Compound 45:N-Acetyl-3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzamide

Acetyl chloride (0.029 mL, 0.41 mmol, 1.17 equiv.) is added to asolution of 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-N-benzamide(Compound A3, 100 mg, 0.35 mmol) in anhydrous tetrahydrofuran (2 mL) andanhydrous pyridine (0.057 mL, 0.705 mmol, 2.00 equiv.) at roomtemperature with stirring, followed by heating of the mixture to 76° C.for 48 h. The reaction mixture is cooled to room temperature,concentrated on a rotary evaporator, and azeotroped once with toluene (2mL portion). The solid is dissolved in dichloromethane (5 mL), washedwith water, dried over MgSO₄ and concentrated to give the crude productas a solid. The product is purified by flash chromatography over a 10 gSiO₂ column (eluted with 5% ethyl acetate/dichloromethane) to give 0.061g (49% yield) ofN-Acetyl-3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzamide (Compound45) as a white solid: mp 119-120° C.; NMR (300 MHz, CDCl₃) δ 2.66 (s,3H), 7.25-7.39 (m, 2H), 7.60-7.71 (m, 2H), 8.05 (d, J=7.8 Hz, 1H), 8.22(t, J=7.2 Hz, 1H), 8.43 (d, J=7.8 Hz, 1H), 8.61 (s, 1H), 8.69 (br s,1H); MS m/z 326.25, calcd for C₁₇H₁₂FN₃O₃ (MH⁺) 326.

D: Compound 47:2-[5-(2-Fluorophenyl)-[1,2,4]oxadiazol-3-yl]-N-(2,2,2-trifluoroacetyl)benzamide

2-[5-(2-Fluorophenyl)-[1,2,4]oxadiazol-3-yl]-N-(2,2,2-trifluoroacetyl)benzamide(Compound 52) may be prepared following the same general procedure asExample C above, except trifluoroacetic anhydride is used in place ofacetyl chloride: mp 119-120° C.; ¹H NMR (300 MHz, CDCl₃) δ 7.26-7.39 (m,2H), 7.62-7.67 (m, 2H), 7.81 (d, J=7.8 Hz, 1H), 8.21 (t, J=7.5 Hz, 1H),8.42 (d, J=8.1 Hz, 1H), 8.49 (s, 1H); MS m/z 361.22 (M-F)⁺ calcd forC₁₇H₉F₄N₃O₃ (MH⁺) 379.

E: Compound 46:3-[5-(2-Fluorophenyl)-[1,2,4]oxadiazol-3-yl]-N-isobutyryl-benzamide (3)

3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]-N-isobutyrylbenzamide(Compound 46) may be prepared following the same general procedure asExample C, except isobutyryl chloride is used in place of acetylchloride: mp 183-184° C.; ¹H NMR (300 MHz, CDCl₃) δ 1.29 (d, J=6.6 Hz,6H), 3.67 (m, 1H), 7.29-7.40 (m, 2H), 7.61-7.71 (m, 2H), 8.06 (d, J=7.8Hz, 1H), 8.26 (t, J=7.8 Hz, 1H), 8.43 (d, J=7.8 Hz, 1H), 8.53 (br s,1H), 8.62 (s, 1H); MS m/z 354.26, calcd for C₁₉H₁₆FN₃O₃ (MH⁺) 354.

Melting point and mass spectrometry (mass spec) data for certainpreferred compounds of the invention are presented in the table below.

Melting Mass Spec Compound Point ° C. Data (ES+) 1 176-177 342.16 2160.5-162   356.2  3 172-174 370.23 4   226-227.5 367.23 5 206-207357.18 6 240 327.17 7 233-234 373.23 8 247-249 405.18 9 252-254 404.2310 252-254 404.16 11 177.5-179   412.20 12 223-224 441.35 13 NT 478.1814 NT 425.25 15 188-191 481.21 16 129-130 432.17 17 172-174 410.16 18134-138 424.19 19 133-134 418.13 20 152-155 370.17 21 139-141 386.11 22187-190 396.18 23 199-202 458.19 24 178-180 381.14 25 190-194 397.15 26161-163 383.18 27 247-250 431.17 28 169-173 472.22 29 NT 429.33 30140-141 430.12 (ES−) 31 150-152 396.13 32 168-170 456.17 (M-H) 33164-165 486.13 34 141-143 400.15 35 197-200 398.1  36 165-168 442.12(M-H) 37 170-173 412.3  38 168-170 398.29 39 108-109 412.3  40 161-163500.24 41 NT 511.33 42 132-134 501.32 43 120-123 439.29 44 193-196399.21 45 119-120 326.25 46 183-184 354.26 47 119-120 361.22 48 180-183433.2  49 152-156 398.23 50 NT 398.23 51 116-118 448.23 (M-H)

Example 2 Nonsense Suppression Activity

A functional, cell-based translation assay based on luciferase-mediatedchemoluminescence (international Application PCT/US2003/023185, filed onJul. 23, 2003, hereby incorporated by reference in its entirety) permitsquantitative assessment of the level of nonsense suppression. Humanembryonic kidney cells (293 cells) are grown in medium containing fetalbovine serum (FBS). These cells can be stably transfected with theluciferase gene containing a premature termination codon at amino acidposition 190. In place of the threonine codon (ACA) normally present inthe luciferase gene at this site, each of the 3 possible nonsense codons(TAA, TAG, or TGA) and each of the 4 possible nucleotides (adenine,thymine, cytosine, or guanine) at the contextually important downstream+1 position following the nonsense codon are introduced by site-directedmutagenesis. As such, amino acid 190 in the luciferase gene containing apremature termination codon is TAA, TAG, or TGA. For each stop codon,the nucleotide following amino acid 190 of luciferase gene containing apremature termination codon can be replaced with an adenine, thymine,cytosine, or guanine (A, T, C, G) such that these mutations do notchange the reading frame of the luciferase gene. Schematics of theseconstructs are depicted in FIG. 1.

The nonsense suppression activity from a cell-based luciferase reporterassay of the present invention as described above shown in the tablebelow (Table 2). Human Embryonic Kidney 293 cells are stably transfectedwith a luciferase reporter construct comprising a UGA nonsense mutationat position 190, which is followed, in-frame by an adenine nucleotide(UGAA).

Activity measurements in Table 2 are determined in a cell-basedluciferase reporter assay of the present invention using a constructcontaining a UGA premature termination codon. Gentamicin, anaminoglycoside antibiotic known to allow readthrough of prematuretermination codons, is used as an internal standard. Activitymeasurements are based on the qualitative ratio between the minimumconcentration of compound required to produce a given protein in a cellversus the amount of protein produced by the cell at that concentration.Compounds which are found to have either or both very high potency andvery high efficacy of protein synthesis are classified as “*****”.Compounds which are found to have intermediate potency and/or efficacyof protein synthesis are classified as “****” “***”; or “**”. Similarly,compounds which are found to have lower potency and/or efficacy ofprotein synthesis are classified as

TABLE 2 Compound UGAA activity 1 ** 2 ** 3 *** 4 * 5 ** 6 * 7 * 8 * 9 *10 ** 11 *** 12 ** 13 *** 14 ** 15 * 16 ** 17 ** 18 ** 19 *** 20 ** 21*** 22 *** 23 ** 24 * 25 ** 26 ** 27 *** 28 * 29 * 30 ** 31 ** 32 ** 33** 34 ** 35 ** 36 ** 37 ** 38 ** 39 ** 40 ** 41 ** 42 *** 43 **** 44**** 45 *** 46 ** 47 ** 48 *** 49 **** 50 **** 51 ***

Nonsense suppression activity in an assay as described above is shown inTable 3 below, for a construct with a UAG nonsense mutation at position190, followed by an adenine nucleotide in-frame, (UAGA); and a constructwith a UAA nonsense mutation at position 190, followed by an adeninenucleotide in-frame, (UAAA).

TABLE 3 Compound No. UAGA activity UAAA activity 43 ** ** 44 ** ** 49 **** 50 ** **

Example 3 Readthrough Assay

A functional, cell-based translation assay based on luciferase-mediatedchemoluminescence (International Application PCT/US2003/023185, filed onJul. 23, 2003 and incorporated by reference in its entirety) permitsassessment of translation-readthrough of the normal stop codon in anmRNA. Human embryonic kidney cells (293 cells) are grown in mediumcontaining fetal bovine serum (FBS). These cells are stably transfectedwith the luciferase gene containing a premature termination codon atamino acid position 190. In place of the threonine codon (ACA) normallypresent in the luciferase gene at this site, each of the 3 possiblenonsense codons (TAA, TAG, or TGA) and each of the 4 possiblenucleotides (adenine, thymine, cytosine, or guanine) at the contextuallyimportant downstream +1 position following the nonsense codon areintroduced by site-directed mutagenesis. As such, amino acid 190 in theluciferase gene containing a premature termination codon is TAA, TAG, orTGA. For each stop codon, the nucleotide following amino acid 190 ofluciferase gene containing a premature termination codon is replacedwith an adenine, thymine, cytosine, or guanine (A, T, C, G) such thatthese mutation do not change the reading frame of the luciferase gene.Schematics of these constructs are depicted above in FIG. 1.

Another assay of the present invention can evaluate compounds thatpromote nonsense mutation suppression. The luciferase constructsdescribed above in FIG. 1 are engineered to harbor two epitope tags inthe N-terminus of the luciferase protein. Based on luciferase proteinproduction, these constructs qualitatively assess the level oftranslation-readthrough. The presence of the full-length luciferaseprotein produced by suppression of the premature termination codon ismeasured by immunoprecipitation of the suppressed luciferase protein(using an antibody against a His tag) followed by Western blotting usingan antibody against the second epitope (the Xpress™ epitope;Invitrogen®; Carlsbad, Calif.). These constructs are depicted in FIG. 2.

Cells that harbor the constructs of FIG. 2 show increased full-lengthprotein production when treated with a compound of the presentinvention. After treatment for 20 hours, cells containing the constructsof FIG. 2 are collected and an antibody recognizing the His epitope isused to immunoprecipitate the luciferase protein. Followingimmunoprecipitation, Western blotting is performed using the antibody tothe Xpress™ epitope (Invitrogen®; Carlsbad, Calif.) to detect thetruncated luciferase (produced when no nonsense suppression occurs) andto detect the full-length protein (produced by suppression of thenonsense codon). Treatment of cells with a test compound producesfull-length protein and not a readthrough protein (See e.g., FIG. 3).The readthrough protein is produced if suppression of the normaltermination codon occurs. Compounds of the present invention suppressthe premature, i.e. nonsense mutation, but not the normal terminationcodon in the luciferase mRNA.

Compounds of the present invention selectively act on prematuretermination codons but not normal termination codons in mammals.

Rats and dogs are administered high doses of compound (up to 1800 mg/kg)by gavage (oral) once daily for 14 days. After the treatment, tissuesare collected, lysates are prepared, and Western blot analysis isperformed. Selection of the proteins for evaluation of normaltermination codon readthrough is based primarily on the correspondingmRNA having a second stop codon in the 3′-UTR that is in-frame with thenominal termination codon. Between these 2 stop codons, each selectedprotein has an intervening sequence of nucleotides that codes for anextension of the protein in the event of ribosomal readthrough of thefirst termination codon. If the compound has the capacity to inducenonspecific, ribosomal readthrough, an elongated protein isdifferentiated from the wild-type protein using Western blot. Tissuesare collected from rats and are analyzed for suppression of the normaltermination codon (UAA) in the vimentin mRNA. No evidence of suppressionis apparent. Tissues are collected from dogs treated with compounds ofthe present invention. There is no evidence of suppression of the normaltermination codon of beta actin, which harbors a UAG stop codon.

In healthy human volunteers, a single dose of a compound of the presentinvention (200 mg/kg) is administered orally. Blood samples arecollected, plasma is prepared, and a Western blot is conducted usingplasma samples from female and male subjects. C-reactive protein (CRP),which harbors a UGA termination codon, is used to determine if treatmentof subjects with compounds of the present invention result insuppression of the normal-termination codon in the CRP mRNA. Aluciferase assay in combination with a premature termination assaydemonstrates selective suppression of premature termination codons butnot normal termination codons.

Example 4 Animal Models

Animal model systems can also be used to demonstrate the safety andefficacy of a compound of the present invention. The compounds of thepresent invention are tested for biological activity using animal modelsfor a disease, condition, or syndrome of interest. These include animalsengineered to contain the target RNA element coupled to a functionalreadout system, such as a transgenic mouse.

Cystic Fibrosis

Examples of animal models for cystic fibrosis include, but are notlimited to, cftr(−/−) mice (see, e.g., Freedman et al., 2001,Gastroenterology 121(4):950-7), cftr(tm1HGU/tm1HGU) mice (see, e.g.,Bernhard et al., 2001, Exp Lung Res 27(4):349-66), CFTR-deficient micewith defective cAMP-mediated Cl(−) conductance (see, e.g., Stotland etal., 2000, Pediatr Pulmonol 30(5):413-24), andC57BL/6-Cftr(m1UNC)/Cftr(m1UNC) knockout mice (see, e.g., Stotland etal., 2000, Pediatr Pulmonol 30(5):413-24).

Muscular Dystrophy

Examples of animal models for muscular dystrophy include, but are notlimited to, mouse, hamster, cat, dog, and C. elegans. Examples of mousemodels for muscular dystrophy include, but are not limited to, the dy−/−mouse (see, e.g., Connolly et al., 2002, J Neuroimmunol 127(1-2):80-7),a muscular dystrophy with myositis (mdm) mouse mutation (see, e.g.,Garvey et al., 2002, Genomics 79(2):146-9), the mdx mouse (see, e.g.,Nakamura et al., 2001, Neuromuscul Disord 11(3):251-9), theutrophin-dystrophin knockout (dko) mouse (see, e.g., Nakamura et al.,2001, Neuromuscul Disord 11(3):251-9), the dy/dy mouse (see, e.g.,Dubowitz et al., 2000, Neuromuscul Disord 10(45):292-8), the mdx(Cv3)mouse model (see, e.g., Pillers et al., 1999, Laryngoscope109(8):1310-2), and the myotonic ADR-MDX mutant mice (see, e.g., Krameret al., 1998, Neuromuscul Disord 8(8):542-50). Examples of hamstermodels for muscular dystrophy include, but are not limited to,sarcoglycan-deficient hamsters (see, e.g., Nakamura et al., 2001, Am JPhysiol Cell Physiol 281(2):C690-9) and the BIO 14.6 dystrophic hamster(see, e.g., Schlenker & Burbach, 1991, J Appl Physiol 71(5):1655-62). Anexample of a feline model for muscular dystrophy includes, but is notlimited to, the hypertrophic feline muscular dystrophy model (see, e.g.,Gaschen & Burgunder, 2001, Acta Neuropathol (Berl) 101(61:591-600).Canine models for muscular dystrophy include, but are not limited to,golden retriever muscular dystrophy (see, e.g., Fletcher et al., 2001,Neuromuscul Disord 11(3):239-43) and canine X-linked muscular dystrophy(see, e.g., Valentine et al., 1992, Am J Med Genet 42(3):352-6).Examples of C. elegans models for muscular dystrophy are described inChamberlain & Benian, 2000, Curr Biol 10(21):R795-7 and Culette &Sattelle, 2000, Hum Mol Genet 9(6):869-77.

Familial Hypercholesterolemia

Examples of animal models for familial hypercholesterolemia include, butare not limited to, mice lacking functional LDL receptor genes (see,e.g., Aji et al., 1997, Circulation 95(2):430-7), Yoshida rats (see,e.g., Fantappie et al., 1992, Life Sci 50(24):1913-24), the JCR:LA-cprat (see, e.g., Richardson et al., 1998, Atherosclerosis 138(1):135-46),swine (see, e.g., Hasler-Rapacz et al., 1998, Am J Med Genet76(5):379-86), and the Watanabe heritable hyperlipidaemic rabbit (see,e.g., Tsutsumi et al., 2000, Arzneimittelforschung 50(2):118-21; Harschet al., 1998, Br J Pharmacol 124(2):227-82; and Tanaka et al., 1995,Atherosclerosis 114(1):73-82).

Human Cancer

An example of an animal model for human cancer, in general includes, butis not limited to, spontaneously occurring tumors of companion animals(see, e.g., Vail & MacEwen, 2000, Cancer Invest 18(8):781-92). Examplesof animal models for lung cancer include, but are not limited to, lungcancer animal models described by Zhang &. Roth (1994, In Vivo8(5):755-69) and a transgenic mouse model with disrupted p53 function(see, e.g., Morris et al., 1998, J La State Med Soc 150(4):179-85). Anexample of an animal model for breast cancer includes, but is notlimited to, a transgenic mouse that overexpresses cyclin Di (see, e.g.,Hosokawa et al., 2001, Transgenic Res 10(5):471-8). An example of ananimal model for colon cancer includes, but is not limited to, a TCRbetaand p53 double knockout mouse (see, e.g., Kado et al., 2001, Cancer Res61(6):2395-8). Examples of animal models for pancreatic cancer include,but are not limited to, a metastatic model of Panc02 murine pancreaticadenocarcinoma (see, e.g., Wang et al., 2001, Int J Pancreatol29(1):37-46) and nu-nu mice generated in subcutaneous pancreatic tumours(see, e.g., Ghaneh et al., 2001, Gene Ther 8(3):199-208). Examples ofanimal models for non-Hodgkin's lymphoma include, but are not limitedto, a severe combined immunodeficiency (“SCID”) mouse (see, e.g., Bryantet al., 2000, Lab Invest 80(4):553-73) and an IgHmu-HOX11 transgenicmouse (see, e.g., Hough et al., 1998, Proc Natl Acad Sci USA95(23):13853-8). An example of an animal model for esophageal cancerincludes, but is not limited to, a mouse transgenic for the humanpapillomavirus type 16 E7 oncogene (see, e.g., Herber et al., 1996, JVirol 70(3):1873-81). Examples of animal models for colorectalcarcinomas include, but are not limited to, Apc mouse models (see, e.g.,Fodde & Smits, 2001, Trends Mol Med 7(8):369-73 and Kuraguchi et al.,2000, Oncogene 19(50):5755-63). An example of an animal model forneurofibromatosis includes, but is not limited to, mutant NF1 mice (see,e.g., Cichowski et al., 1996, Semin Cancer Biol 7(5):291-8). Examples ofanimal models for retinoblastoma include, but are not limited to,transgenic mice that expression the simian virus 40 T antigen in theretina (see, e.g., Howes et al., 1994, Invest Ophthalmol Vis Sci35(2):342-51 and Windle et al, 1990, Nature 343(62591:665-9) and inbredrats (see, e.g., Nishida et al., 1981, Curr Eye Res 1(1):53-5 andKobayashi et al., 1982, Acta Neuropathol (Berl) 57(2-3):203-8). Examplesof animal models for Wilm's tumor include, but are not limited to, a WT1knockout mice (see, e.g., Scharnhorst et al., 1997, Cell Growth Differ8(4133-43), a rat subline with a high incidence of neuphroblastoma (see,e.g., Mesfin & Breech, 1996, Lab Anim Sci 46(3):321-6), and aWistar/Furth rat with Wilms' tumor (see, e.g., Murphy et al., 1987,Anticancer Res 7(4B):717-9).

Retinitis Pigmentosa

Examples of animal models for retinitis pigmentosa include, but are notlimited to, the Royal College of Surgeons (“RCS”) rat (see, e.g.,Vollrath et al., 2001, Proc Natl Acad Sci USA 98(22); 12584-9 andHanitzsch et al., 1998, Acta Anat (Basel) 162(2-3):119-26), a rhodopsinknockout mouse (see, e.g., Jaissle et al., 2001, Invest Ophthalmol VisSci 42(2):506-13), and Wag/Rij rats (see, e.g., Lai et al., 1980, Am JPathol 98(1):281-4).

Cirrhosis

Examples of animal models for cirrhosis include, but are not limited to,CCl₄-exposed rats (see, e.g., Kloehn et al., 2001, Horm Metab Res33(7):394-401) and rodent models instigated by bacterial cell componentsor colitis (see, e.g., Vierling, 2001, Best Pract Res Clin Gastroenterol15(4):591-610).

Hemophilia

Examples of animal models for hemophilia include, but are not limitedto, rodent models for hemophilia A (see, e.g., Reipert et al., 2000,Thromb Haemost 84(5):826-32; Jarvis et al., 1996, Thromb Haemost75(2):318-25; and Bi et al., 1995, Nat Genet 10(1):119-21), caninemodels for hemophilia A (see, e.g., Gallo-Perm et al., 1999, Hum GeneTher 10(11):1791-802 and Connelly et al, 1998, Blood 91(9); 3273-81),murine models for hemophilia B (see, e.g., Snyder et al., 1999, Nat Med5(1):64-70; Wang et al., 1997, Proc Natl Acad Sci USA 94(21):11563-6;and Fang et al., 1996, Gene Ther 3(3):217-22), canine models forhemophilia B (see, e.g., Mount et al., 2002, Blood 99(8):2670-6; Snyderet al., 1999, Nat Med 5(1):64-70; Fang et al., 1996, Gene Ther3(3):217-22); and Kay et al., 1994, Proc Natl Acad Sci USA91(6):2353-7), and a rhesus macaque model for hemophilia B (see, e.g.,Lozier et al., 1999, Blood 93(6):1875-81).

von Willebrand Disease

Examples of animal models for von Willebrand disease include, but arenot limited to, an inbred mouse strain RIIIS/J (see, e.g., Nichols etal., 1994, 83(11):3225-31 and Sweeney et al., 1990, 76(10:2258-65), ratsinjected with botrocetin (see, e.g., Sanders et al., 1988, Lab Invest59(4):443-52), and porcine models for von Willebrand disease (see, e.g.,Nichols et al., 1995, Proc Natl Acad Sci USA 92(7):2455-9; Johnson &Bowie, 1992, J Lab Clin Med 120(4):553-8); and Brinkhous et al., 1991,Mayo Clin Proc 66(7):733-42).

β-Thalassemia

Examples of animal models for β-thalassemia include, but are not limitedto, murine models with mutations in globin genes (see, e.g., Lewis etal., 1998, Blood 91(6):2152-6; Raja et al., 1994, Br J Haematol86(1):156-62; Popp et al., 1985, 445:432-44; and Skow et al., 1983, Cell34(41043-52).

Kidney Stones

Examples of animal models for kidney stones include, but are not limitedto, genetic hypercalciuric rats (see, e.g., Bushinsky et al., 1999,Kidney Int 55(1):234-43 and Bushinsky et al., 1995, Kidney Int48(6):1705-13), chemically treated rats (see, e.g., Grases et al., 1998,Scand J Urol Nephrol 32(4):261-5; Burgess et al., 1995, Urol Res23(4):239-42, Kumar et al., 1991, J Urol 146(5):1384-9; Okada et al.,1985, Hinyokika Kiyo 31(4):565-77; and Bluestone et al., 1975, LabInvest 33(3):273-9), hyperoxaluric rats (see, e.g., Jones et al., 1991,J Urol 145(4):868-74), pigs with unilateral retrograde flexiblenephroscopy (see, e.g., Seifmah et al., 2001, 57(4):832-6), and rabbitswith an obstructed upper urinary tract (see, e.g., Itatani et al., 1979,Invest Urol 17(3):234-40).

Ataxia-Telangiectasia

Examples of animal models for ataxia-telangiectasia include, but are notlimited to, murine models of ataxia-telangiectasia (see, e.g., Barlow etal., 1999, Proc Acad Sci USA 96(17):9915-9 and Inoue et al., 1986,Cancer Res 46(8):3979-82).

Lysosomal Storage Diseases

Examples of animal models for lysosomal storage diseases include, butare not limited to, mouse models for mucopolysaccharidosis type VII(see, e.g., Brooks et al., 2002, Proc Natl Acad Sci USA. 99(9):6216-21;Monroy et al., 2002, Bone 30(2):352-9; Vogler et al., 2001, Pediatr DevPathol. 4(5):421-33; Vogler et al., 2001, Pediatr Res. 49(3):342-8; andWolfe et al., 2000, Mol Ther. 2(6):552-6), a mouse model formetachromatic leukodystrophy (see, e.g., Matzner et al., 2002, GeneTher. 9(1):51-63), a mouse model of Sandhoff disease (see, e.g., Sangoet al., 2002, Neuropathol Appl Neurobiol. 28(1):23-34), mouse models formucopolysaccharidosis type III A (see, e.g., Bhattacharyya et al., 2001,Glycobiology 11(1):99-10 and Bhaumik et al., 1999, Glycobiology9(12):1389-96.), arylsulfatase A (ASA)-deficient mice (see, e.g.,D'Hooge et al., 1999, Brain Res. 847(2):352-6 and D'Hooge et al, 1999,Neurosci Lett. 273(2):93-6); mice with an aspartylglucosaminuriamutation (see, e.g., Jalanko et al., 1998, Hum Mol Genet. 7(2):265-72);feline models of mucopolysaccharidosis type VI (see, e.g., Crawley etal., 1998, J Clin Invest. 101(1):109-19 and Norrdin et al., 1995, Bone17(5):485-9); a feline model of Niemann-Pick disease type C (see, e.g.,March et al., 1997, Acta Neuropathol (Berl), 94(2):164-72); acidsphingomyelinase-deficient mice (see, e.g., Otterbach & Stoffel, 1995,Cell 81(7):1053-6), and bovine mannosidosis (see, e.g., Jolly et al.,1975, Birth Defects Orig Arctic Ser. 11(6):273-8).

Tuberous Sclerosis

Examples of animal models for tuberous sclerosis (“TSC”) include, butare not limited to, a mouse model of TSC1. (see, e.g., Kwiatkowski etal., 2002, Hum Mol Genet. 11(5):525-34), a Tsc1 (TSC1 homologue)knockout mouse (see, e.g., Kobayashi et al., 2001, Proc Natl Acad SciUSA. 2001 Jul. 17; 98(15):8762-7), a TSC2 gene mutant (Eker) rat model(see, e.g., Hino 2000, Nippon Rinsho 58(6):1255-61; Mizuguchi et al.,2000, J Neuropathol Exp Neurol. 59(3):188-9; and Hino et al., 1999, ProgExp Tumor Res. 35:95-108); and Tsc2(+/−) mice (see, e.g., Onda et al.,1999, J Clin Invest. 104(6):687-95).

Example 5 mdx Mouse, an Animal Model Study

The mutation in the mdx mouse that causes premature translationtermination of the 427 kDa dystrophin polypeptide has been shown to be aC to T transition at position 3185 in exon 23 (Sicinski et al., Science244(4912):1578-1580 (1989)), Mouse primary skeletal muscle culturesderived from 1-day old mdx mice are prepared as described previously(Barton-Davis et al., J. Clin. Invest, 104(4):375-381 (1999)). Cells arecultured for 10 days in the presence of a compound of the invention.Culture medium is replaced every four days and the presence ofdystrophin in myoblast cultures is detected by immunostaining asdescribed previously (Barton-Davis et al., J. Clin. Invest.104(4):375-381 (1999), hereby incorporated by reference in itsentirety). A primary monoclonal antibody to the C-terminus of thedystrophin protein is used undiluted and rhodamine conjugated anti-mouseIgG is used as the secondary antibody. The antibody detects thefull-length protein produced by suppression of the nonsense codon.Staining is viewed using a Leica DMR microscope, digital camera, andassociated imaging software.

As previously described (Barton-Davis et al., J. Clin. Invest.104(4):375-381 (1999), a compound is delivered by Alzet osmotic pumpsimplanted under the skin of anesthetized mice. Two doses of a compoundof the invention are administered. Gentamicin serves as a positivecontrol and pumps filled with solvent serve as the negative control.Pumps are loaded with appropriate compound such that the calculateddoses to which tissue is exposed are 10 mM and 20 mM. The gentamicinconcentration is calculated to achieve tissue exposure of approximately200 mM. In the initial experiment, mice are treated for 14 days, afterwhich animals are anesthetized with ketamine and exsanguinated. Thetibialis anterior (TA) muscle of the experimental animals is thenexcised, frozen, and used for immunofluorescence analysis of dystrophinincorporation into striated muscle. The presence of dystrophin in TAmuscles is detected by immunostaining, as described previously(Barton-Davis et al., J. Clin. Invest. 104(4):375-381 (1999).

Western Blot Analysis

Quadricep muscles from an mdx mouse treated with a compound of thepresent invention for 4 weeks are analyzed by Western blot using acommercially available antibody to dystrophin. Protein extracted fromthe quadriceps of a wild-type mouse serve as a positive control.Production of full-length dystrophin is observed in the treated animal.The amount of full-length dystrophin produced, as a result of nonsensesuppression, but not limited by this theory, is approximately 10% ofwild-type levels of expression.

Immunofluorescence

Male mdx mice (age 9-11 weeks) are treated with different compounds ofthe present invention (n=2 at least for each compound). These compoundsare injected SQ once per day for two weeks at 25 mg/kg. After 2 weeks oftreatment, mice are sacrificed for the removal of muscles to determinedystrophin readthrough efficiency.

Immunofluorescence (IF) is performed on 10 μm cryosections using adystrophin antibody. The antibody recognizes an epitope C-terminal tothe premature stop mutation found in mdx mice. Image analysis isperformed in an identical manner in all sections. Images from treatedand untreated mice are analyzed and a signal greater than the signal onthe untreated control is deemed positive and indicates that suppressionof the premature termination codon in the dystrophin mRNA occurred.

Muscle Mechanics

Isolated whole muscle mechanics is performed on EDL muscles fromanimals. Optimum muscle length (Lo) is defined as the length thatproduced maximum twitch tension. Maximum tetanic force at Lo is measuredusing a 120 Hz, 500 msec pulse at supramaximal voltage. Protectionagainst mechanical injury, induced by a series of 5 eccentric tetaniccontractions, is monitored. These measurements are performed using a 700msec stimulation period during which the muscle is held in an isometriccontraction for the first 500 msec followed by a stretch of 8 or 10% Loat a rate of 0.5 Lo/sec. Protection against mechanical injury isevaluated at 80 Hz stimulation frequency. Damage is determined as theloss in force between the first and last eccentric contraction.Treatment with compounds of the present invention result in protectionfrom damage induced by eccentric contractions of the EDL muscle comparedto the untreated control.

Example 6 Suppression of a Nonsense Mutation in the p53 Gene

For an animal model system, CAOV-3 cells (1×10⁷) are injected into theflanks of nude/nude mice. After 12 days, mice are randomized (10 miceper group) and treated subcutaneously (5 days per week) with 3 mg/kg ofa compound of the present invention or intraperitonealy (1 day per week)with 30 mg/kg of a compound of the present invention. Tumor volumes aremeasured weekly. Suppression of nonsense mutations in the p53 gene by acompound of the present invention can inhibit cancer growth in vivo.

Example 7 Access to Specific Nucleotides of the 28S rRNA is Modified byCompounds of the Present Invention

Previous studies have demonstrated that gentamicin and other members ofthe aminoglycoside family that decrease the fidelity of translation bindto the A site of the 16S rRNA. By chemical footprinting, UVcross-linking and NMR, gentamicin has been shown to bind at the A site(comprised of nucleotides 1400-1410 and 1490-1500, E. coli numbering) ofthe rRNA at nucleotides 1406, 1407, 1494, and 1496 (Moazed & Noller,Nature 327(6121):389-394 (1978); Woodcock et al., EMBO J.10(10):3099-3103 (1991); and Schroeder et al., EMBO J. 19:1-9 (2000).

Ribosomes prepared from HeLa cells are incubated with the smallmolecules (at a concentration of 100 mM), followed by treatment withchemical modifying agents (dimethyl sulfate [DMS] and kethoxal [KE]).Following chemical modification, rRNA is phenol-chlorofolin extracted,ethanol precipitated, analyzed in primer extension reactions usingend-labeled oligonucleotides hybridizing to different regions of thethree rRNA and resolved on 6% polyacrylamide gels. Probes for primerextension cover the entire 18S (7 oligonucleotide primers), 28S (24oligonucleotide primers), and 5S (one primer) rRNAs. Controls in theseexperiments include DMSO (a control for changes in rRNA accessibilityinduced by DMSO), paromomycin (a marker for 18S rRNA binding), andanisomycin (a marker for 28S rRNA binding).

All publications and patent applications cited herein are incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

Although certain embodiments have been described in detail above, thosehaving ordinary skill in the art will clearly understand that manymodifications are possible in the embodiments without departing from theteachings thereof. All such modifications are intended to be encompassedwithin the claims of the invention.

What is claimed is:
 1. A method of treating Duchenne muscular dystrophyassociated with a nonsense mutation in the dystrophin gene in a patientin need thereof, wherein the nonsense mutation results in a prematuretermination codon in mRNA, wherein the premature termination codonresults in either or both premature mRNA translation termination ornonsense-mediated mRNA decay, comprising administering to the patient aneffective amount of a compound selected from the group consisting of:

and a pharmaceutically acceptable salt, racemate or stereoisomerthereof.